Selective induction of apoptosis to treat ocular disease

ABSTRACT

The invention is directed to a method of prophylactically or therapeutically treating choroidal neovascularization, wherein the method comprises directly administering to the eye a therapeutic factor or a nucleic acid sequence that encodes a therapeutic factor, which he expressed to produce the therapeutic factor, to selectively induce apoptosis of endothelial cells associated with neovascularization of the choroid such that choroidal neovascularization is treated prophylactically or therapeutically. The invention also provides a method of prophylactically or therapeutically treating ocular neovascularization, wherein the method comprises directly administering to the eye a nucleic acid sequence encoding a therapeutic factor to promote apoptosis of endothelial cells associated with neovascularization, such that the nucleic acid is expressed thereby producing the therapeutic factor to treat ocular neovascularization prophylactically or therapeutically.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of copending U.S. patentapplication Ser. No. 10/367,038, filed Feb. 14, 2003, which claims thebenefit of U.S. Provisional Patent Application No. 60/357,340, filedFeb. 15, 2002.

FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made in part with Government support under GrantNumbers EYO5951, EYO12609, and K08 awarded by the National EyeInstitute, National Institutes of Health. The Government may havecertain rights in this invention.

FIELD OF THE INVENTION

The invention relates to a method of prophylactically or therapeuticallytreating an ocular disorder associated with abnormal neovascularization.

BACKGROUND OF THE INVENTION

An overwhelming majority of the world's population will experience somedegree of vision loss in their lifetime. Vision loss affects virtuallyall people regardless of age, race, economic or social status, orgeographical location. Ocular-related disorders, while often not lifethreatening, necessitate life-style changes that jeopardize theindependence of the afflicted individual. Vision impairment can resultfrom most all ocular disorders, including diabetic retinopathies,proliferative retinopathies, retinal detachment, toxic retinopathies,retinal vascular diseases, retinal degenerations, vascular anomalies,age-related macular degeneration and other acquired disorders,infectious diseases, inflammatory diseases, ocular ischemia,pregnancy-related disorders, retinal tumors, choroidal tumors, choroidaldisorders, vitreous disorders, trauma, cataract complications, dry eye,and inflammatory optic neuropathies.

Leading causes of severe vision loss and blindness are ocular-relateddisorders wherein the vasculature of the eye is damaged orinsufficiently regulated. Ocular-related diseases comprising aneovascularization complication are many and include, for example,exudative age-related macular degeneration, diabetic retinopathy,corneal neovascularization, choroidal neovascularization, cyclitis,Hippel-Lindau Disease, retinopathy of prematurity, pterygium,histoplasmosis, iris neovascularization, macular edema,glaucoma-associated neovascularization, and the like. It is likely thatsevere vision loss does not result directly from neovascularization, butthe effect of neovascularization on the retina. The retina is a delicateocular membrane on which images are received. Near the center of theretina is the macula lutea, an oval area of retinal tissue where visualsense is most acute. The retina is most delicate at the fovea centralis,the central depression located in the center of the macula. Damage ofthe retina, i.e., retinal detachment, retinal tears, or retinaldegeneration, is directly connected to vision loss. A common cause ofretinal detachment, retinal tears, and retinal degeneration is abnormal,i.e., uncontrolled, vascularization of various ocular tissues, althougha small percentage of cases are due to atrophic complications. Disordersassociated with both neovascular and atrophic components, such asage-related macular degeneration and diabetic retinopathy, areparticularly difficult to treat due to the emergence of a wide varietyof complications.

Age-related macular degeneration (AMD) is the leading cause of blindnessin patients over 65 years of age. As the elderly population of the worldincreases, the incidence of age-related macular degeneration is expectedto increase dramatically, reaching a predicted 7.5 million cases in theUnited States alone by the year 2030 (Hyman et al., Am. J. Epidemiol.,118, 213-227 (1983)). Age-related macular degeneration is a progressive,degenerative disorder of the eye resulting initially in loss of visualacuity. Many patients afflicted with AMD experience exudativecomplications, including disciform scars (i.e., scarring involvingfibrous elements) and neovascularization. Severe vision loss occurs asneovascularization or atrophy disturbs the foveal center (Bressler etal., Ophthalmology, 102, 1206-1211 (1995)). Ultimately, legal blindnessfrom age-related macular degeneration stems from degeneration of the RPEand the subsequent death of photoreceptors.

Like AMD, diabetic retinopathy is subdivided into a nonproliferativestage, which typically occurs first, and a proliferative stage. Theproliferative stage of diabetic retinopathy is characterized byneovascularization and fibrovascular growth (i.e., scarring involvingglial and fibrous elements) from the retina or optic nerve over theinner surface of the retina or disc or into the vitreous cavity.

Abnormal vascularization of the eye also can occur in the layer directlyunderneath the retina, i.e., the choroid. Choroidal neovascularization(CNV) is often associated with AMD and results in leakage, bleeding, andscarring in the macula. Scarring in the macula results in a centralscotoma (interruption of the visual field) and loss of reading anddriving vision. Choroidal neovascularization is detected usingangiography, e.g., fluorescein angiography, alone or in combination withindocyanine-green angiography.

For many ocular-related disorders, there are currently no effectivetherapeutic options. Laser photocoagulation, the administration of laserburns to various areas of the eye, is used in the treatment of manyneovascularization-linked disorders. For example, focal macularphotocoagulation is used to treat areas of vascular leakage in themacula (Murphy, Amer. Family Physician, 51(4), 785-796 (1995)).Similarly, neovascularization, in particular, advanced proliferativeretinopathy, is commonly treated with scatter or panretinalphotocoagulation. However, laser treatment may cause permanent blindspots corresponding to the treated areas. With respect to age-relatedmacular degeneration, many patients eventually experience severe visionloss in spite of treatment. Other treatment options for ocular-relateddisorders include thermotherapy, photodynamic therapy, radiationtherapy, and surgery to either translocate the macula or remove theabnormal blood vessels. However, only photodynamic therapy and focallaser have been found to be better than no treatment at all, and thetreatment effect is marginal and temporary.

Given the prevalence of ocular-related disorders and the lack ofeffective treatments, there remains a need for an effective prophylacticand therapeutic treatment of ocular-related disorders, in particularocular-related disorders with complications associated with abnormalcellular proliferation (e.g., neovascularization), such as diabeticretinopathy, age-related macular degeneration, and choroidalneovascularization. Accordingly, the invention provides materials andmethods for prophylactically and therapeutically treating disordersassociated with neovascularization, in particular, ocular-relateddisorders associated with neovascularization. This and other advantagesof the invention will become apparent from the detailed descriptionprovided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method for the prophylactic or therapeutictreatment of ocular-related disorders. In particular, the inventionprovides a method of prophylactically or therapeutically treatingneovascularization in the eye, e.g., choroidal neovascularization. Themethod comprises directly administering a therapeutic factor, oradministering a nucleic acid sequence encoding a therapeutic factor,which is expressed to produce the therapeutic factor, to the eye toselectively induce apoptosis of endothelial cells associated withneovascularization, thereby treating the ocular disease prophylacticallyor therapeutically. Preferably, the therapeutic factor is an inhibitorof angiogenesis or a neurotrophic agent. More preferably, thetherapeutic factor comprises both anti-angiogenic and neurotrophicactivity, such as PEDF.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to a method of prophylactically ortherapeutically treating an animal, preferably a mammal (e.g., a human),for at least one ocular-related disorder associated withneovascularization. Ocular-related disorders appropriate for treatmentusing the inventive method include, but are not limited to, diabeticretinopathies, proliferative retinopathies, retinopathy of prematurity,retinal vascular diseases, vascular anomalies, age-related maculardegeneration and other acquired disorders, endophthalmitis, infectiousdiseases, inflammatory diseases, AIDS-related disorders, ocular ischemiasyndrome, pregnancy-related neovascular disorders, peripheral retinaldegenerations, retinal degenerations with neovascular complications,toxic retinopathies, retinal tumors, corneal neovascularization,choroidal tumors, choroidal disorders, choroidal neovascularization,neovascular glaucoma, vitreous disorders, retinal detachment andproliferative vitreoretinopathy associated with neovascularization,macular edema, iris neovascularization, neovascularization associatedwith severe myopia, surgical-induced neovascular disorders, and thelike, which are not mutually exclusive.

In particular, the invention provides a method of prophylactically ortherapeutically treating an animal for ocular neovascularization. Themethod comprises directly administering a therapeutic factor or anucleic acid sequence encoding a therapeutic factor to the eye toselectively induce apoptosis of endothelial cells associated withneovascularization. When the method comprises contacting an ocular cellwith a nucleic acid sequence encoding the therapeutic factor, thenucleic acid sequence is expressed to produce the therapeutic factor toprophylactically or therapeutically treat ocular neovascularization. By“directly administering to the eye” is meant that the nucleic acidsequence contacts cells on, within, and surrounding the globe of theeye. In other words, the nucleic acid sequence can contact cellsassociated with the globe of the eye, such as, but not limited to, cellson the surface of the eye (e.g., conjunctival cells), or cells that makeup the inner layers of the eye (e.g., cells of the retinal or choriodallayers). The nucleic acid sequence also can contact cells of the ocularapparatus such as, but not limited to, ocular muscle cells, cells liningthe ocular orbital, fibroblasts, and the like. The therapeutic factor isproduced in the cells of the ocular apparatus and penetrates the globeof the eye to exert a biological effect on endothelial cells, directlyor indirectly. Desirably, the therapeutic factor is an inhibitor ofangiogenesis (e.g., via apoptosis of appropriate cells) or aneurotrophic agent. Most preferably, the therapeutic factor comprisesboth anti-angiogenic and neurotrophic activities.

The inventive method is superior over previously described methods oftreating neovascularization via destruction of the cells associated withneovascularization in that the inventive method selectively killsendothelial cells associated with new vascular structures, therebyminimizing harm to existing vasculature within the eye. Whileneovascularization throughout the body has common features, each area ofneovascularization also comprises unique characteristics. Indeed, it hasbeen proven that neovascular processes in different vascular beds of thesame organ show overlapping, but not identical characteristics. Inaddition, it has been demonstrated that newly formed blood vessels andestablished blood vessels differ with respect to factors required forpersistence of the vessels (Campochiaro, J. Cell. Phys., 184, 301-310(2000)). Thus, it is possible to capitalize on the distinctions betweenneovascular processes and established blood vessels to selectivelyablate cells associated with new blood vessel growth, therebyprophylactically or therapeutically treating ocular disorders associatedwith neovascularization. The therapeutic factor of the inventive methodselectively induces apoptosis of cells associated withneovascularization compared to cells associated with existingvasculature. Desirably, apoptosis is selectively induced in endothelialcells associated with uncontrolled or abnormal angiogenesis. By“selectively inducing apoptosis” is meant that apoptosis induced by thetherapeutic factor in cells associated with neovascularization is atleast about five times greater than apoptosis induced by the therapeuticfactor in cells associated with existing vasculature. More preferably,the level of apoptosis in cells associated with neovascularization(e.g., endothelial cells) induced by the therapeutic factor is at leastabout 5-times greater, preferably at least about 10-times greater (e.g.,at least about 15-times greater, at least about 20-times greater, atleast about 30-times greater) than the level of apoptosis induced by thetherapeutic factor in cells associated with existing vasculature. Evenmore preferably, apoptosis in neovascular cells is at least about50-times greater than the level of apoptosis in cells of existingvasculature when apoptosis is induced by the therapeutic factor. Mostpreferably, the therapeutic factor does not induce apoptosis in cells ofexisting vasculature (i.e., blood vessels present before the onset ofdiseased neovascularization).

The ocular neovascularization treated by the inventive method can beneovascularization associated with any region of the eye. Preferably theneovascularization is neovascularization of the choroid. The choroid isa thin, vascular membrane located under the retina. Abnormalneovascularization of the choroid results from, for example, age-relatedmacular degeneration, histoplasmosis, myopic degeneration, angioidstreaks, choroidal rupture, photocoagulation, or any disease thatresults in a choroidal scar or break in Bruch's membrane such as, forexample, Best's disease, choroidal hemangioma, metallic intraocularforeign body, choroidal nonperfusion, choroidal osteomas, bacterialendocarditis, choroideremia, chronic retinal detachment, drusen, depositof metabolic waste material, endogenous Candida endophthalmitis,neovascularization at ora serrata, operating microscope burn, punctateinner choroidopathy, radiation retinopathy, retinal cryoinjury,retinitis pigmentosa, retinochoroidal coloboma, rubella, subretinalfluid drainage, tilted disc syndrome, Toxoplasma retinochoroiditis,tuberculosis, and the like. When the inventive method is used to treator prevent choroidal neovascularization, apoptosis of endothelial cellsof the neovasculature of the choroid is sought, with little, if any,apoptosis of endothelial cells associated with existing vasculature ofthe choroid or the retina.

Neovascularization of the cornea is also appropriate for treatment bythe method of the invention. The cornea is a projecting, transparentsection of the fibrous tunic, which is the outer most layer of the eye.The outermost layer of the cornea contacts the conjunctiva, while theinnermost layer comprises the endothelium of the anterior chamber.Corneal neovascularization stems from, for example, ocular injury,surgery, infection, improper wearing of contact lenses, and diseasessuch as, for example, corneal dystrophies.

Alternatively, the ocular neovascularization is neovascularization ofthe retina. Retinal neovascularization is an indication associated withnumerous ocular diseases and disorders, many of which are named above.Preferably, the neovascularization of the retina treated in accordancewith the inventive method is associated with diabetic retinopathy.Common causes of retinal neovascularization include ischemia, viralinfection, and retinal damage. Neovascularization of the retina can leadto macular edema, subretinal discoloration, scarring, and the like.Complications associated with retina neovascularization stem frombreakage and leakage of newly formed blood vessels. Vision is impairedas blood fills the vitreous cavity and is not efficiently removed. Notonly is the passage of light impeded, but an inflammatory response tothe excess blood and metabolites can cause further damage to oculartissue. Thus, the inventive method can protect against macular edema byinhibiting the formation of leaky diseased neovasculature. In addition,the new vessels form fibrous scar tissue, which, over time, will disturbthe retina causing retinal tears and detachment. In addition, vision isimpaired by the subsequent damage or destruction of photoreceptors.

The method of the invention also is useful in prophylactically ortherapeutically treating an animal for age-related macular degenerationassociated with at least one exudative complication. Exudativecomplications include, for example, disciform scars (i.e., scarringinvolving fibrous elements) and neovascularization. Prophylactic andtherapeutic treatment of age-related macular degeneration is furtherdiscussed in, for example, International Patent Application WO 01/58494.

An embodiment of the invention provides a method for prophylactically ortherapeutically treating an animal for choroidal neovascularization. Themethod comprises directly administering to the eye a therapeutic factoror a nucleic acid sequence encoding the therapeutic factor thatselectively induces apoptosis of endothelial cells associated withchoroidal neovascularization. Preferably, the existing vascularizationof the eye is not affected. In that a great deal of damage occurs as aresult of edema, thickening of underlying membranes, and build-up ofmetabolic byproducts, preferably the nucleic acid sequence encoding thetherapeutic factor (or the therapeutic factor itself) is administered toan area of vascular leakage or an area adjacent to vascular leakage.

By “prophylactic” is meant the protection, in whole or in part, againstocular neovascularization. By “therapeutic” is meant the amelioration ofocular neovascularization, itself, and, desirably, the protection, inwhole or in part, against further ocular neovascularization. One ofordinary skill in the art will appreciate that any degree of protectionfrom, or amelioration of, ocular neovascularization is beneficial to apatient. The invention is particularly advantageous in that thetherapeutic factor is directly applied to affected areas without some ofthe harmful side effects of many presently employed therapies.

The inventive method is useful in the treatment of both acute andpersistent, progressive ocular neovascularization. For acute ailments,the therapeutic factor or the nucleic acid sequence encoding thetherapeutic factor can be administered using a single or multipleapplications within a short time period. If neovascularization persists,numerous applications of the therapeutic factor may be necessary torealize a therapeutic effect.

The therapeutic factor, which in most cases will comprise a protein orpeptide with therapeutic activity, can be delivered to the eye in apharmaceutically-acceptable carrier. Ideally, the therapeutic factor isadministered directly to the eye to minimize transmission of thetherapeutic factor to non-target tissues. Alternatively, a nucleic acidsequence encoding the therapeutic factor can be delivered to the eyewhere it is expressed. The produced therapeutic factor is therebyadministered to target ocular cells.

Preferably, a nucleic acid sequence encoding the therapeutic factor isdirectly administered to the eye. Ideally, the nucleic acid sequence isincorporated into an expression vector. Any of a number of expressionvectors known in the art and able to transduce ocular cells is suitablefor use in the inventive methods. Examples of suitable expressionvectors include, for instance, plasmids, plasmid-liposome complexes, andviral vectors, e.g., parvoviral-based vectors (i.e., adeno-associatedvirus (AAV)-based vectors), retroviral vectors, herpes simplex virus(HSV)-based vectors, AAV-adenoviral chimeric vectors, andadenovirus-based vectors. These expression vectors can be prepared usingstandard recombinant DNA techniques described in, e.g., Sambrook et al.,Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1989), and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, New York, N.Y. (1994).

Plasmids, genetically engineered circular double-stranded DNA molecules,can be designed to contain an expression cassette for delivery of thenucleic acid sequence encoding at least one inhibitor of angiogenesisand/or at least one neurotrophic factor to an ocular cell. Althoughplasmids were the first vector described for administration oftherapeutic nucleic acids, the level of transfection efficiency is poorcompared with other techniques. By complexing the plasmid withliposomes, the efficiency of gene transfer in general is improved. Whilethe liposomes used for plasmid-mediated gene transfer strategies havevarious compositions, they are typically synthetic cationic lipids.Advantages of plasmid-liposome complexes include their ability totransfer large pieces of DNA encoding a therapeutic nucleic acid andtheir relatively low immunogenicity. Plasmids can be complexed withprotein moieties to target specific cell surface receptors, if desired.

Plasmids are often used for short-term expression. However, a plasmidconstruct can be modified to obtain prolonged expression. It hasrecently been discovered that the inverted terminal repeats (ITR) ofparvovirus, in particular adeno-associated virus (AAV), are responsiblefor the high-level persistent nucleic acid expression often associatedwith AAV (see, for example, U.S. Pat. No. 6,165,754). Accordingly, theexpression vector can be a plasmid comprising native parvovirus ITRs toobtain prolonged and substantial expression of the therapeutic factor.While plasmids are suitable for use in the inventive method, preferablythe expression vector is a viral vector.

AAV vectors are viral vectors of particular interest for use in genetherapy protocols. AAV is a DNA virus, which is not known to cause humandisease. AAV requires co-infection with a helper virus (i.e., anadenovirus or a herpes virus), or expression of helper genes, forefficient replication. AAV vectors used for administration of atherapeutic nucleic acid have approximately 96% of the parental genomedeleted, such that only the ITRs, which contain recognition signals forDNA replication and packaging, remain. This eliminates immunologic ortoxic side effects due to expression of viral genes. In addition,delivering the AAV rep protein enables integration of the AAV vectorcomprising AAV ITRs into a specific region of genome, if desired. Hostcells comprising an integrated AAV genome show no change in cell growthor morphology (see, for example, U.S. Pat. No. 4,797,368). Althoughefficient, the need for helper virus or helper genes can be an obstaclefor widespread use of this vector.

Retrovirus is an RNA virus capable of infecting a wide variety of hostcells. Upon infection, the retroviral genome integrates into the genomeof its host cell and is replicated along with host cell DNA, therebyconstantly producing viral RNA and any nucleic acid sequenceincorporated into the retroviral genome. When employing pathogenicretroviruses, e.g., human immunodeficiency virus (HIV) or human T-celllymphotrophic viruses (HTLV), care must be taken in altering the viralgenome to eliminate toxicity. A retroviral vector can additionally bemanipulated to render the virus replication-incompetent. As such,retroviral vectors are thought to be particularly useful for stable genetransfer in vivo. Lentiviral vectors, such as HIV-based vectors, areexemplary of retroviral vectors used for gene delivery.

HSV-based viral vectors are suitable for use as an expression vector tointroduce nucleic acids into ocular cells. The mature HSV virionconsists of an enveloped icosahedral capsid with a viral genomeconsisting of a linear double-stranded DNA molecule that is 152 kb. Mostreplication-deficient HSV vectors contain a deletion to remove one ormore intermediate-early genes to prevent replication. The advantages ofthe herpes vector are its ability to enter a latent stage that canresult in long-term DNA expression, and its large viral DNA genome thatcan accommodate exogenous DNA up to 25 kb. Of course, this ability isalso a disadvantage in terms of short-term treatment regimens. For adescription of HSV-based vectors appropriate for use in the inventivemethod, see, for example, U.S. Pat. Nos. 5,837,532; 5,846,782;5,849,572; and 5,804,413 and International Patent Applications WO91/02788, WO 96/04394, WO 98/15637, and WO 99/06583.

Adenovirus (Ad) is a 36 kb double-stranded DNA virus that efficientlytransfers DNA in vivo to a variety of different target cell types. Foruse in the inventive method, the virus is preferably made replicationdeficient by deleting select genes required for viral replication. Theexpendable E3 region is also frequently deleted to allow additional roomfor a larger DNA insert. The vector can be produced in high titers andcan efficiently transfer DNA to replicating and non-replicating cells.The newly transferred genetic information remains epi-chromosomal, thuseliminating the risks of random insertional mutagenesis and permanentalteration of the genotype of the target cell. However, if desired, theintegrative properties of AAV can be conferred to adenovirus byconstructing an AAV-Ad chimeric vector. For example, the AAV ITRs andnucleic acid encoding the Rep protein incorporated into an adenoviralvector enables the adenoviral vector to integrate into a mammalian cellgenome. Therefore, AAV-Ad chimeric vectors are an interesting option foruse in the invention. Similarly, conditionally replication-competentadenoviral vectors, wherein gene functions required for viralreplication are encoded by the vector but expressed only in response tocertain stimuli, can be used to deliver the nucleic acid sequenceencoding the therapeutic factor to target cells. Such vectors aredescribed in, for example, U.S. Pat. No. 5,998,205.

Preferably, the nucleic acid sequence encoding the therapeutic factor isincorporated into a viral vector; more preferably, the nucleic acidsequence encoding the therapeutic factor is present in an adenoviralvector. In the context of the invention, the adenoviral vector can bederived from, for example, any serotype of human adenovirus. Adenoviralstocks that can be employed as a source of adenovirus can be amplifiedfrom the adenoviral serotypes 1 through 51, which are currentlyavailable from the American Type Culture Collection (ATCC, Manassas,Va.), or from any other serotype of adenovirus available from any othersource. For instance, an adenovirus can be of subgroup A (e.g.,serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16,21, 34, and 35), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D(e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41), orany other adenoviral serotype. Preferably, however, an adenovirus is ofserotype 2, 5 or 9. However, non-group C adenoviruses can be used toprepare replication-deficient adenoviral gene transfer vectors fordelivery of one or more therapeutic factors to ocular cells. Preferredadenoviruses used in the construction of non-group C adenoviral genetransfer vectors include Ad12 (group A), Ad7 (group B), Ad30 and Ad36(group D), Ad4 (group E), and Ad41 (group F). Non-group C adenoviralvectors, methods of producing non-group C adenoviral vectors, andmethods of using non-group C adenoviral vectors are disclosed in, forexample, U.S. Pat. Nos. 5,801,030; 5,837,511; and 5,849,561 andInternational Patent Applications WO 97/12986 and WO 98/53087.

The adenoviral vector is preferably deficient in at least onereplication-essential gene function (i.e., a gene function required forviral replication), thereby resulting in a “replication-deficient”adenoviral vector. Preferably, the adenoviral vector is deficient in atleast one replication-essential gene function of the E1 region of theadenoviral genome (i.e., a gene function required for viralreplication). In addition to a deficiency in the E1 region, therecombinant adenovirus can also have a mutation in the major latepromoter (MLP). The mutation in the MLP can be in any of the MLP controlelements such that it alters the responsiveness of the promoter, asdiscussed in International Patent Application WO 00/00628. Morepreferably, the vector is deficient in at least onereplication-essential gene function of the E1 region and at least partof the E3 region (e.g., an Xba I deletion of the E3 region). Withrespect to the E1 region, the adenoviral vector can be deficient in atleast part of the E1a region and/or at least part of the E1b region.

Preferably, the adenoviral vector is “multiply-deficient,” meaning thatthe adenoviral vector is deficient in one or more replication-essentialgene functions in each of two or more regions, i.e., the E1, E2, E4, L1,L2, L3, L4, and/or L5 regions. For example, the aforementionedE1-deficient or E1-, E3-deficient adenoviral vectors can be furtherdeficient in at least one replication-essential gene function of the E4region. Adenoviral vectors deleted of the entire E4 region can elicitlower host immune responses.

Alternatively, the adenoviral vector lacks all or part of the E1 regionand all or part of the E2 region. However, adenoviral vectors lackingall or part of the E1 region, all or part of the E2 region, and all orpart of the E3 region also are contemplated herein. In one embodiment,the adenoviral vector lacks all or part of the E1 region, all or part ofthe E2 region, all or part of the E3 region, and all or part of the E4region. Suitable replication-deficient adenoviral vectors are disclosedin U.S. Pat. Nos. 5,851,806 and 5,994,106 and International PatentApplications WO 95/34671 and WO 97/21826. For example, suitablereplication-deficient adenoviral vectors include those with at least apartial deletion of the E1a region, at least a partial deletion of theE1b region, at least a partial deletion of the E2a region, and at leasta partial deletion of the E3 region. Alternatively, thereplication-deficient adenoviral vector can have at least a partialdeletion of the E1 region, at least a partial deletion of the E3 region,and at least a partial deletion of the E4 region. Suchmultiply-deficient viral vectors are particularly useful in that suchvectors can accept large inserts of exogenous DNA. Indeed, adenoviralamplicons, an example of a multiply-deficient adenoviral vector whichcomprises only those genomic sequences required for packaging andreplication of the viral genome (e.g., at least one inverted terminalrepeat (ITR) and packaging signal), can accept inserts of approximately36 kb.

Therefore, in a preferred embodiment, the expression vector of theinventive method is a multiply-deficient adenoviral vector lacking allor part of the E1 region, all or part of the E3 region, all or part ofthe E4 region, and, optionally, all or part of the E2 region. In thisregard, it has been observed that an at least E4-deficient adenoviralvector expresses a transgene at high levels for a limited amount of timein vivo and that persistence of expression of a transgene in an at leastE4-deficient adenoviral vector can be modulated through the action of atrans-acting factor, such as HSV ICP0, Ad pTP, CMV-IE2, CMV-IE86, HIVtat, HTLV-tax, HBV-X, AAV Rep 78, the cellular factor from the U205osteosarcoma cell line that functions like HSV ICP0, or the cellularfactor in PC12 cells that is induced by nerve growth factor, amongothers. In view of the above, the multiply-deficient adenoviral vector(e.g., the at least E4-deficient adenoviral vector) preferably furthercomprises a nucleic acid sequence encoding a trans-acting factor thatmodulates the persistence of expression of the nucleic acid sequenceencoding the therapeutic factor. Alternatively, a second nucleic acidsequence encoding a trans-acting factor that modulates the persistenceof expression of the nucleic acid sequence encoding the therapeuticfactor is administered to the eye. Preferably, the nucleic acid sequenceencoding the trans-acting factor does not encode an adenoviral E4 regiongene product. Whether expressed from the adenoviral vector or suppliedby a second expression vector, preferably, the trans-acting factor isthe Herpes simplex infected cell polypeptide 0 (HSV ICP0).

It should be appreciated that the deletion of different regions of aviral vector can alter the immune response of the mammal. For example,deletion of different regions can reduce the inflammatory responsegenerated by the adenoviral vector. Furthermore, the adenoviral vector'scoat protein can be modified so as to decrease the adenoviral vector'sability or inability to be recognized by a neutralizing antibodydirected against the wild-type coat protein, as described inInternational Patent Application WO 98/40509. Such modifications areuseful for long-term treatment of persistent ocular neovascularization.

Similarly, the coat protein of a viral vector, preferably an adenoviralvector, can be manipulated to alter the binding specificity orrecognition of a virus for a viral receptor on a potential host cell.For adenovirus, such manipulations can include deletion of regions ofthe fiber, penton, hexon, pIIIa, pVI, and pIX, insertions of variousnative or nonnative ligands into portions of the coat protein, and thelike. Manipulation of the coat protein can broaden the range of cellsinfected by a viral vector or enable targeting of a viral vector to aspecific cell type. For example, in one embodiment, the expressionvector is a viral vector comprising a chimeric coat protein (e.g., afiber, hexon, pIX, pIIIa, or penton protein), which differs from thewild-type (i.e., native) coat protein by the introduction of a nonnativeamino acid sequence, preferably at or near the carboxyl terminus.Preferably, the nonnative amino acid sequence is inserted into or inplace of a coat protein sequence. The nonnative amino acid sequence canbe inserted within the internal coat protein sequence or at the end ofthe coat protein sequence. The resultant chimeric viral coat protein isable to direct entry into cells of the viral, i.e., adenoviral, vectorcomprising the coat protein that is more efficient than entry into cellsof a vector that is identical except for comprising a wild-type viralcoat protein rather than the chimeric viral coat protein. Preferably,the chimeric virus coat protein binds a novel endogenous binding sitepresent on the cell surface that is not recognized, or is poorlyrecognized by a vector comprising a wild-type coat protein. One directresult of this increased efficiency of entry is that the virus,preferably, the adenovirus, can bind to and enter cell types which avirus comprising wild-type coat protein typically cannot enter or canenter with only a low efficiency.

In another embodiment of the invention, the nucleic acid sequence ispresent in a viral vector comprising a chimeric virus coat protein notselective for a specific type of eukaryotic cell. The chimeric coatprotein differs from the wild-type coat protein by an insertion of anonnative amino acid sequence into or in place of an internal coatprotein sequence. In this embodiment, the chimeric virus coat proteinefficiently binds to a broader range of eukaryotic cells than awild-type virus coat, such as described in International PatentApplication WO 97/20051.

Specificity of binding of an adenovirus to a given cell can also beadjusted by use of an adenovirus comprising a short-shafted adenoviralfiber gene, as discussed in U.S. Pat. No. 5,962,311. Use of anadenovirus comprising a short-shafted adenoviral fiber gene reduces thelevel or efficiency of adenoviral fiber binding to its cell-surfacereceptor and increases adenoviral penton base binding to itscell-surface receptor, thereby increasing the specificity of binding ofthe adenovirus to a given cell. Alternatively, use of an adenoviruscomprising a short-shafted fiber enables targeting of the adenovirus toa desired cell-surface receptor by the introduction of a nonnative aminoacid sequence either into the penton base or the fiber knob.

In addition, the coat protein of a viral vector, in particular anadenoviral vector, can be manipulated to ablate native binding of thecoat protein to cell surface receptors. Ablation of native binding ofthe adenoviral coat proteins, e.g., ablation of those amino acidsequences in the fiber and penton associated with binding to thecoxsackie and adenovirus receptor (CAR) and integrins, respectively, canbe advantageous in generating targeted vectors.

Of course, the ability of a viral vector to recognize a potential hostcell can be modulated without genetic manipulation of the coat protein.For instance, complexing an adenovirus with a bispecific moleculecomprising a penton base-binding domain and a domain that selectivelybinds a particular cell surface binding site enables one of ordinaryskill in the art to target the vector to a particular cell type.

Suitable modifications to a viral vector, specifically an adenoviralvector, are described in U.S. Pat. Nos. 5,559,099; 5,731,190; 5,712,136;5,770,442; 5,846,782; 5,926,311; 5,965,541; 6,057,155; 6,127,525;6,153,435; 6,329,190; and 6,455,314 and International PatentApplications WO 96/07734, WO 96/26281, WO 97/20051, WO 98/07865, WO98/07877, WO 98/54346, WO 00/15823, WO 01/58940, and WO 01/92549.Similarly, it will be appreciated that numerous expression vectors areavailable commercially. Construction of expression vectors is wellunderstood in the art. Adenoviral vectors can be constructed and/orpurified using the methods set forth, for example, in U.S. Pat. No.5,965,358 and International Patent Applications WO 98/56937, WO99/15686, and WO 99/54441. Adeno-associated viral vectors can beconstructed and/or purified using the methods set forth, for example, inU.S. Pat. No. 4,797,368 and Laughlin et al., Gene, 23, 65-73 (1983).

The selection of an expression (i.e., delivery) vector for use in theinventive method to administer the nucleic acid sequence encoding thetherapeutic factor will depend on a variety of factors such as, forinstance, the host, immunogenicity of the vector, the desired durationof protein production, and the like. As each type of expression vectorhas distinct properties, a researcher has the freedom to tailor theinventive method to any particular situation. Moreover, more than onetype of expression vector (i.e., a plasmid and a viral vector or twodifferent viral vectors) can be used to deliver the nucleic acidsequence to the ocular cell. Thus, the invention can comprise directlyadministering to the eye, thereby contacting an ocular cell, withdifferent expression vectors, each comprising the nucleic acid sequenceencoding the therapeutic factor. The nucleic acid sequence encoding thetherapeutic factor is expressed, thereby resulting in the production ofthe therapeutic factor to prophylactically or therapeutically treatocular neovascularization in an animal. If multiple types of expressionvectors are used, preferably an adenoviral vector and anadeno-associated viral vector are directly administered to the eye. Oneof ordinary skill in the art will appreciate the ability to capitalizeon the advantageous properties of multiple delivery systems to treat orstudy ocular neovascularization.

The nucleic acid sequence encoding the therapeutic factor is desirablyoperably linked to regulatory sequences necessary for expression, e.g.,a promoter. A “promoter” is a DNA sequence that directs the binding ofRNA polymerase and thereby promotes RNA synthesis. A nucleic acidsequence is “operably linked” to a promoter when the promoter is capableof directing transcription of that nucleic acid sequence. A promoter canbe native or nonnative to the nucleic acid sequence to which it isoperably linked.

Any promoter (i.e., whether isolated from nature or produced byrecombinant DNA or synthetic techniques) can be used in connection withthe invention to provide for transcription of the nucleic acid sequence.The promoter preferably is capable of directing transcription in aeukaryotic (desirably mammalian) cell. The functioning of the promotercan be altered by the presence of one or more enhancers and/or silencerspresent on the vector. “Enhancers” are cis-acting elements of DNA thatstimulate or inhibit transcription of adjacent genes. An enhancer thatinhibits transcription also is termed a “silencer.” Enhancers differfrom DNA-binding sites for sequence-specific DNA binding proteins foundonly in the promoter (which also are termed “promoter elements”) in thatenhancers can function in either orientation, and over distances of upto several kilobase pairs (kb), even from a position downstream of atranscribed region.

A comparison of promoter sequences that function in eukaryotes hasrevealed conserved sequence elements. Generally, eukaryotic promoterstranscribed by RNA polymerase II are typified by a “TATA box” centeredat approximately position −25, which appears to be essential foraccurately positioning the start of transcription. The TATA box directsRNA polymerase to begin transcribing approximately 30 base pairs (bp)downstream in mammalian systems. The TATA box functions in conjunctionwith at least two other upstream sequences located about 40 bp and 110bp upstream of the start of transcription. Typically, a so-called “CCAATbox” serves as one of the two upstream sequences, and the other often isa GC-rich segment. The CCAAT homology can reside on different strands ofthe DNA. The upstream promoter element also can be a specialized signalsuch as one of those which have been described in the art and whichappear to characterize a certain subset of genes.

To initiate transcription, the TATA box and the upstream sequences areeach recognized by regulatory proteins which bind to these sites, andactivate transcription by enabling RNA polymerase II to bind the DNAsegment and properly initiate transcription. Whereas base changesoutside the TATA box and the upstream sequences have little effect onlevels of transcription, base changes in either of these elementssubstantially lower transcription rates (see, e.g., Myers et al.,Science, 229, 242-247 (1985); McKnight et al., Science, 217, 316-324(1982)). The position and orientation of these elements relative to oneanother, and to the start site, are important for the efficienttranscription of some, but not all, coding sequences. For instance, somepromoters function well in the absence of any TATA box. Similarly, thenecessity of these and other sequences for promoters recognized by RNApolymerase I or III, or other RNA polymerases, can differ.

Accordingly, promoter regions can vary in length and sequence and canfurther encompass one or more DNA binding sites for sequence-specificDNA binding proteins and/or an enhancer or silencer. Enhancers and/orsilencers can similarly be present on a nucleic acid sequence outside ofthe promoter per se.

The invention preferentially employs a viral promoter. Suitable viralpromoters are known in the art and include, for instance,cytomegalovirus (CMV) promoters, such as the CMV immediate-earlypromoter, promoters derived from human immunodeficiency virus (HIV),such as the HIV long terminal repeat promoter, Rous sarcoma virus (RSV)promoters, such as the RSV long terminal repeat, mouse mammary tumorvirus (MMTV) promoters, HSV promoters, such as the Lap2 promoter or theherpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci.,78, 144-145 (1981)), promoters derived from SV40 or Epstein Barr virus,adeno-associated viral promoters, such as the p5 promoter, and the like.Preferably, the viral promoter is an adenoviral promoter, such as theAd2 or Ad5 major late promoter and tripartite leader, a CMV promoter, oran RSV promoter.

Many of the above-described promoters are constitutive promoters.Instead of being a constitutive promoter, the promoter can be aninducible promoter, i.e., a promoter that is up- and/or down-regulatedin response to appropriate signals. For instance, the regulatorysequences can comprise a hypoxia driven promoter, which is active whenthe ocular neovascularization is associated with hypoxia. Other examplesof suitable inducible promoter systems include, but are not limited to,the IL-8 promoter, the metallothionine inducible promoter system, thebacterial lacZYA expression system, the tetracycline expression system,and the T7 polymerase system. Further, promoters that are selectivelyactivated at different developmental stages (e.g., globin genes aredifferentially transcribed from globin-associated promoters in embryosand adults) can be employed. The promoter sequence that regulatesexpression of the nucleic acid sequence can contain at least oneheterologous regulatory sequence responsive to regulation by anexogenous agent. The regulatory sequences are preferably responsive toan exogenous agent such as, but not limited to, a drug, a hormone, oranother gene product. For example, the regulatory sequence, e.g.,promoter, preferably is responsive to a glucocorticoid receptor-hormonecomplex, which, in turn, enhances the level of transcription of atherapeutic peptide or a therapeutic fragment thereof.

Preferably, the regulatory sequence comprises a tissue-specificpromoter, i.e., a promoter that is preferentially activated in a giventissue and results in expression of a gene product in the tissue whereactivated. A typically used tissue-specific promoter is amyocyte-specific promoter. A tissue specific promoter for use in theinventive vector can be chosen by the ordinarily skilled artisan basedupon the target tissue or cell-type. Preferred tissue-specific promotersfor use in the inventive methods are specific to ocular tissue, such asa rhodopsin promoter. Examples of rhodopsin promoters include, but arenot limited to, a GNAT cone-transducing alpha-subunit gene promoter oran interphotoreceptor retinoid binding protein promoter.

One of ordinary skill in the art will appreciate that each promoterdrives transcription, and, therefore, protein expression, differentlywith respect to time and the amount of protein produced. For example,the CMV promoter is characterized as having peak activity shortly aftertransduction, i.e., about 24 hours after transduction, then quicklytapering off. On the other hand, the RSV promoter's activity increasesgradually, reaching peak activity several days after transduction, andmaintains a high level of activity for several weeks. Indeed, sustainedexpression driven by an RSV promoter has been observed in all cell typesstudied, including, for instance, liver cells, lung cells, spleen cells,diaphragm cells, skeletal muscle cells, and cardiac muscle cells. Thus,a promoter can be selected for use in the method of the invention bymatching its particular pattern of activity with the desired pattern andlevel of expression of at least one inhibitor of angiogenesis and/or atleast one neurotrophic factor. Alternatively, a hybrid promoter can beconstructed which combines the desirable aspects of multiple promoters.For example, a CMV-RSV hybrid promoter combining the CMV promoter'sinitial rush of activity with the RSV promoter's high maintenance levelof activity would be especially preferred for use in many embodiments ofthe inventive method. It is also possible to select a promoter with anexpression profile that can be manipulated by an investigator.

Also preferably, the expression vector comprises a nucleic acid encodinga cis-acting factor, wherein the cis-acting factor modulates theexpression of the nucleic acid sequence. Preferably, the cis-actingfactor comprises matrix attachment region (MAR) sequences (e.g.,immunoglobulin heavy chain (Jenunwin et al., Nature, 385(16), 269(1997)), apolipoprotein B, or locus control region (LCR) sequences,among others. MAR sequences have been characterized as DNA sequencesthat associate with the nuclear matrix after a combination of nucleasedigestion and extraction (Bode et al., Science, 255(5041), 195-197(1992)). MAR sequences are often associated with enhancer-typeregulatory regions and, when integrated into genomic DNA, MAR sequencesaugment transcriptional activity of adjacent nucleotide sequences. Ithas been postulated that MAR sequences play a role in controlling thetopological state of chromatin structures, thereby facilitating theformation of transcriptionally-active complexes. Similarly, it isbelieved LCR sequences function to establish and/or maintain domainspermissive for transcription. Many LCR sequences give tissue specificexpression of associated nucleic acid sequences. Addition of MAR or LCRsequences to the expression vector can further enhance expression of atleast one inhibitor of angiogenesis and/or at least one neurotrophicfactor.

With respect to promoters, nucleic acid sequences, selectable markers,and the like, located on an expression vector comprising the nucleicacid sequence encoding the therapeutic factor according to theinvention, such elements can be present as part of a cassette, eitherindependently or coupled. In the context of the invention, a “cassette”is a particular base sequence that possesses functions which facilitatesubcloning and recovery of nucleic acid sequences (e.g., one or morerestriction sites) or expression (e.g., polyadenylation or splice sites)of particular nucleic acid sequences.

The construction of an exogenous nucleic acid operably linked toregulatory sequences necessary for expression is well within the skillof the art (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2d ed. (1989)). With respect to the expression of thenucleic acid sequence encoding the therapeutic factor (as well as othernucleic acid sequences) according to the invention, the ordinary skilledartisan is aware that different genetic signals and processing eventscontrol levels of nucleic acids and proteins/peptides in a cell, suchas, for example, transcription, mRNA translation, andpost-transcriptional processing. The transcription of DNA into RNArequires a functional promoter, as described herein.

Protein expression is dependent on the level of RNA transcription thatis regulated by DNA signals, and the levels of DNA template. Similarly,translation of mRNA requires, at the very least, an AUG initiationcodon, which is usually located within 10 to 100 nucleotides of the 5′end of the message. Sequences flanking the AUG initiator codon have beenshown to influence its recognition by eukaryotic ribosomes, withconformity to a perfect Kozak consensus sequence resulting in optimaltranslation (see, e.g., Kozak, J. Molec. Biol., 196, 947-950 (1987)).Also, successful expression of an exogenous nucleic acid in a cell canrequire post-translational modification of a resultant protein. Thus,production of a protein can be affected by the efficiency with which DNA(or RNA) is transcribed into mRNA, the efficiency with which mRNA istranslated into protein, and the ability of the cell to carry outpost-translational modification. These are all factors of which theordinary skilled artisan is aware and is capable of manipulating usingstandard means to achieve the desired end result.

Along these lines, to optimize protein production, preferably thenucleic acid sequence further comprises a polyadenylation site followingthe coding region of the nucleic acid sequence. Also, preferably all theproper transcription signals (and translation signals, whereappropriate) are correctly arranged such that the nucleic acid sequencewill be properly expressed in the cells into which it is introduced. Ifdesired, the nucleic acid sequence also can incorporate splice sites(i.e., splice acceptor and splice donor sites) to facilitate mRNAproduction. Moreover, if the nucleic acid sequence encodes a protein orpeptide, which is a processed or secreted protein or actsintracellularly, preferably the nucleic acid sequence further comprisesthe appropriate sequences for processing, secretion, intracellularlocalization, and the like.

In certain embodiments, it may be advantageous to modulate production ofthe therapeutic factor. An especially preferred method of modulatingexpression of a nucleic acid sequence comprises addition ofsite-specific recombination sites on the expression vector. Contacting anucleic acid sequence comprising site-specific recombination sites witha recombinase will either up- or down-regulate transcription of a codingsequence, or simultaneously up-regulate transcription of one codingsequence and down-regulate transcription of another, through therecombination event. Use of site-specific recombination to modulatetranscription of a nucleic acid sequence is described in, for example,U.S. Pat. Nos. 5,801,030 and 6,063,627 and International PatentApplication WO 97/09439.

Preferably, the therapeutic factor that selectively promotes apoptosisof endothelial cells associated with neovascularization is an inhibitorof angiogenesis, e.g., the therapeutic factor induces apoptosis in cellsassociated with neovascularization. When a nucleic acid sequence isadministered, the nucleic acid sequence preferably encodes an inhibitorof angiogenesis that induces apoptosis of endothelial cells associatedwith neovascularization to a greater degree than endothelial cellsassociated with existing vasculature. Multiple therapeutic factors,e.g., inhibitors of angiogenesis, can be administered to the eye. By“inhibitor of angiogenesis” is meant any factor that prevents orameliorates neovascularization. Examples of suitable inhibitors ofangiogenesis that selectively induce apoptosis of endothelial cellsassociated with neovascularization include, but are not limited to, PEDFand endostatin. One of ordinary skill in the art will understand thatcomplete prevention or amelioration of neovascularization is notrequired in order to realize a therapeutic effect. Therefore, theinventive method contemplates both partial and complete prevention andamelioration of angiogenesis. One of ordinary skill in the art willappreciate that the therapeutic factor can be modified or truncated andretain activity.

Endostatin is a carboxy-terminal peptide of collagen XVIII. Theanti-angiogenic peptide has been demonstrated to inhibit endothelialcell proliferation, induce apoptosis of endothelial cells in vitro(Dhanabal et al., J. Biol. Chem., 274(17), 11721-11726 (1999)), andreduce tumor size in mice (Chen et al., Human Gene Therapy, 11,1983-1996 (2000)). The endostatin protein is relatively nontoxic, andbiologically-relevant amounts of the protein have been produced in vivousing a variety of gene transfer vectors.

The therapeutic factor that selectively induces apoptosis in endothelialcells associated with neovascularization also can have neurotrophicactivity (e.g., neurotrophic factor or neurotrophic agent). Neurotrophicfactors are thought to be responsible for the maturation of developingneurons and for maintaining adult neurons and, therefore, are useful formaintaining the viability or prolonging survival of photoreceptor cells.Neurotrophic factors are divided into three subclasses: neuropoieticcytokines; neurotrophins; and fibroblast growth factors. Ciliaryneurotrophic factor (CNTF) is exemplary of neuropoietic cytokines. CNTFpromotes the survival of ciliary ganglionic neurons and supports certainneurons that are NGF-responsive. Neurotrophins include, for example,brain-derived neurotrophic factor and nerve growth factor, perhaps thebest characterized neurotrophic factor. Other neurotrophic factorssuitable for being encoded by the nucleic acid sequence of the inventivemethod includes, for example, transforming growth factors, glialcell-line derived neurotrophic factor, neurotrophin 3, neurotrophin ⅘,and interleukin 1-β. Neurotrophic factors associated with angiogenesis,such as aFGF and bFGF, are less preferred. The neurotrophic factor ofthe inventive method also can be a neuronotrophic factor, e.g., a factorthat enhances neuronal survival. It has been postulated thatneurotrophic factors can actually reverse degradation of neurons. Suchfactors, conceivably, are useful in treating the degeneration of neuronsassociated with vision loss and caused by neovascularization.Neurotrophic factors function in both paracrine and autocrine fashions,making them ideal therapeutic agents. Preferably, the therapeutic factorcomprises both anti-angiogenic activity and neurotrophic activity. Mostpreferably, the therapeutic factor is pigment epithelium-derived factor(PEDF).

PEDF, also named early population doubling factor-1 (EPC-1), is asecreted protein having homology to a family of serine proteaseinhibitors named serpins. PEDF is made predominantly by retinal pigmentepithelial cells and is detectable in most tissues and cell types of thebody. PEDF has been observed to induce differentiation in retinoblastomacells and enhance survival of neuronal populations (Chader, CellDifferent., 20, 209-216 (1987)). Factors that enhance neuronal survivalunder adverse conditions, such as PEDF, are termed “neuronotrophic,” asdescribed herein. PEDF further has gliastatic activity, i.e., theability to inhibit glial cell growth. As discussed above, PEDF also hasanti-angiogenic activity. Anti-angiogenic derivatives of PEDF includeSLED proteins, discussed in International Patent Application WO99/04806. It has also been postulated that PEDF is involved with cellsenescence (Pignolo et al., J. Biol. Chem., 268(12), 8949-8957 (1998)).PEDF for use in the inventive method can be derived from any source, andis further characterized in U.S. Pat. No. 5,840,686 and InternationalPatent Applications WO 93/24529 and WO 99/04806.

The therapeutic factor or the nucleic acid sequence encoding thetherapeutic factor, e.g., PEDF, can be obtained from any source, e.g.,isolated from nature, synthetically or recombinantly generated, isolatedfrom a genetically engineered organism, and the like. In nature, PEDF isalmost solely generated in human fetus retinal cells. The poorproduction of human PEDF from RPE cells and the scarcity of sourcetissue of PEDF complicates the use of this potentially valuabletherapeutic factor. A viral vector comprising the nucleic acid sequenceencoding PEDF can be used to create sufficient amounts of recombinantPEDF protein in cell culture, or can be directly administered to the eyeto produce recombinant PEDF protein intraocularly or extraocularly(e.g., to produce recombinant PEDF in the ocular orbit or cells of theocular apparatus).

Active fragments of the therapeutic factor (i.e., those fragments havingbiological activity sufficient to induce apoptosis of endothelial cellsassociated with neovascularization) are suitable for use in theinventive method. Likewise, a fusion protein comprising the therapeuticfactor or a therapeutic fragment thereof and for example, a moiety thatstabilizes peptide conformation, also can be used. The ordinarilyskilled artisan has the ability to determine whether a modifiedtherapeutic factor or a fragment thereof has the ability to selectivelyinduce apoptosis in endothelial cells associated with neovascularizationusing, for example, the TdT-dUTP terminal nick end-labeling (TUNEL)assay in conjunction with models of angiogenesis, such as in vitroangiogenesis assays, (e.g., Matrigel-based assays), the mouse ear modelof neovascularization, and the rat hindlimb ischemia model. Ideally, theangiogenesis model will involve neovascularization of the eye, such astransgenic mice comprising an exogenous VEGF gene operably linked to therhodopsin promoter, which provides an ocular angiogenesis model in whichneovascularization sprouts from the retinal capillary bed and invadesthe photoreceptor layer and subretinal space (see, for example, Okamatoet al., Am. J. Pathol, 151, 281-291 (1997), or Tobe et al., Invest.Ophthalmol. Vis. Sci., 39, 180-188 (1998)). Alternatively, disruption ofBruch's membrane in the eyes of mice or rabbits provides a reliablemodel of CNV (see, for example, Tobe et al., Am. J. Pathol., 153,1641-1646 (1998)).

The invention also contemplates the use of nucleic acid sequencesencoding chimeric or fusion peptides in the inventive method. Throughrecombinant DNA technology, scientists have been able to generate fusionproteins that contain the combined amino acid sequence of two or moreproteins. The ordinarily skilled artisan can fuse the active domains oftwo or more factors to generate chimeric peptides with desired activity.The chimeric peptide can comprise the entire amino acid sequences of twoor more peptides or, alternatively, can be constructed to compriseportions of two or more peptides (e.g., 10, 20, 50, 75, 100, 400, 500,or more amino acid residues). Desirably, the chimeric peptide comprisesanti-angiogenic and neurotrophic activity, which can be determined usingroutine methods.

Additional agents can be administered in conjunction with thetherapeutic factor or nucleic acid sequence encoding the therapeuticfactor. For example, additional therapeutic peptides, such asanti-inflammatory peptides, immune suppressors, anti-angiogenic factors,or neurotrophic factors, are administered before, during, or afteradministration of the therapeutic factor that selectively inducesapoptosis in endothelial cells associated with neovascularization.

The method of the invention can be part of a treatment regimen involvingother therapeutic modalities. Accordingly, the ocularneovascularization, e.g., choroidal neovascularization or retinalneovascularization, can be treated in accordance with the inventivemethod prior to, during, or after treatment with any of a number ofocular therapies, such as drug therapy, photodynamic therapy,photocoagulation laser therapy, panretinal therapy, thermotherapy,radiation therapy, or surgery. Preferably, the surgery is removal ofsubretinal blood or removal of subretinal choroidal neovascularmembrane. For example, the nucleic acid sequence encoding thetherapeutic factor (or the therapeutic factor itself) can beadministered intraocularly or periocularly (e.g., sub-tenon delivery)for the prophylactic or therapeutic treatment of persistent or recurrentocular neovascularization treated with surgery, laser photocoagulation,and photodynamic therapies.

The therapeutic factor or nucleic acid sequence encoding the therapeuticfactor is preferably administered as soon as possible after it has beendetermined that an animal, such as a mammal, specifically a human, is atrisk for ocular neovascularization (prophylactic treatment) or has begunto develop ocular neovascularization (therapeutic treatment). Treatmentwill depend, in part, upon the particular therapeutic factor used, theparticular nucleic acid sequence used (if appropriate), the route ofadministration, and the cause and extent, if any, of ocularneovascularization realized. For example, systemic administration oradministration to both eyes is preferred in the prophylactic treatmentof neovascularization associated with macular degeneration because, onceone eye is affected, the other eye is at risk (up to 19% per year).

The therapeutic factor or nucleic acid sequence encoding the therapeuticfactor desirably is administered in a pharmaceutical composition (e.g.,a pharmaceutical composition comprising a pharmaceutically acceptablecarrier and the nucleic acid sequence encoding the therapeutic factor).Any suitable pharmaceutically acceptable carrier (e.g.,pharmacologically or physiologically acceptable carrier) can be usedwithin the context of the invention, and such carriers are well known inthe art. The choice of carrier will be determined, in part, by theparticular site to which the composition is to be administered and theparticular method used to administer the composition.

Suitable formulations include aqueous and non-aqueous solutions,isotonic sterile solutions, which can contain anti-oxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood or intraocular fluid of the intended recipient, and aqueous andnon-aqueous sterile suspensions that can include suspending agents,solubilizers, thickening agents, stabilizers, and preservatives. Theformulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, immediately prior to use.Extemporaneous solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described. Whenadministering a nucleic acid sequence or peptide, preferably thepharmaceutically acceptable carrier is a buffered saline solution. Morepreferably, the nucleic acid sequence or peptide is administered in apharmaceutical composition formulated to protect the nucleic acid orpeptide sequence from damage prior to administration. For example, thepharmaceutical composition can be formulated to reduce loss of thenucleic acid sequence or peptide on devices used to prepare, store, oradminister the nucleic acid sequence or therapeutic factor, such asglassware, syringes, or needles. The pharmaceutical composition can beformulated to decrease the light sensitivity and/or temperaturesensitivity of the nucleic acid sequence or the therapeutic factoritself. To this end, the pharmaceutical composition preferably comprisesa pharmaceutically acceptable liquid carrier, such as, for example,those described above, and a stabilizing agent selected from the groupconsisting of polysorbate 80, L-arginine, polyvinylpyrrolidone,trehalose, and combinations thereof. The use of such a pharmaceuticalcomposition will extend the shelf life of the therapeutic factor ornucleic acid sequence encoding the therapeutic factor, facilitateadministration, and increase the efficiency of the inventive method. Inthis regard, a pharmaceutical composition also can be formulated toenhance transduction efficiency. See, for example, U.S. Pat. No.6,225,289 and International Patent Application WO 00/34444 for adiscussion of formulations suitable for pharmaceutical compositions.

In addition, one of ordinary skill in the art will appreciate that thetherapeutic factor or the nucleic acid sequence encoding the therapeuticfactor, e.g., a viral vector comprising the nucleic acid sequence, canbe present in a composition with other therapeutic orbiologically-active agents. For example, therapeutic factors useful inthe treatment of a particular indication can be present. For instance,if treating vision loss, hyaluronidase can be added to a composition toaffect the break down of blood and blood proteins in the vitreous of theeye. Factors that control inflammation, such as ibuprofen or steroids,can be part of the composition to reduce swelling and inflammationassociated with in vivo administration of the viral vector and oculardistress. Immune system suppressors can be administered in combinationwith the inventive method to reduce any immune response to the vectoritself or associated with an ocular disorder. Anti-angiogenic factors,such as soluble growth factor receptors, growth factor antagonists,i.e., angiotensin, and the like also can be part of the composition, aswell as additional neurotrophic factors. Similarly, vitamins andminerals, anti-oxidants, and micronutrients can be co-administered.Antibiotics, i.e., microbicides and fungicides, can be present to reducethe risk of infection associated with gene transfer procedures and otherdisorders.

Suitable methods, i.e., invasive and noninvasive methods, of directlyadministering a therapeutic factor or a nucleic acid sequence encoding atherapeutic factor, whereon the therapeutic factor or nucleic acidsequence will contact an ocular cell, are available. By “directadministration” is meant introduction of the therapeutic factor ornucleic acid sequence encoding the therapeutic factor to the eye.Although more than one route can be used for direct administration, aparticular route can provide a more immediate and more effectivereaction than another route. Accordingly, the described routes ofadministration are merely exemplary and are in no way limiting.

The inventive method is not dependent on the mode of administering thetherapeutic factor or nucleic acid sequence encoding the therapeuticfactor to an animal, preferably a human, to achieve the desired effect.As such, any route of administration is appropriate so long as thetherapeutic factor is administered directly to the eye and contacts anendothelial cell associated with neovascularization. The therapeuticfactor or nucleic acid sequence encoding the therapeutic factor can beappropriately formulated and administered in the form of an injection,eye lotion, ointment, implant and the like. For instance, an expressionvector comprising the nucleic acid sequence of the inventive method canbe applied, for example, topically, subconjunctivally, intraocularly,retrobulbarly, periocularly (e.g., via sub-tenon injection),subretinally, or suprachoroidally. In certain cases, it may beappropriate to administer multiple applications and employ multipleroutes, e.g., subretinal and intravitreous, to ensure sufficientexposure of ocular cells to the therapeutic factor or nucleic acidsequence encoding the therapeutic factor to achieve the desired effect.

Depending on the particular case, it may be desirable to non-invasivelyadminister the therapeutic factor or nucleic acid sequence encoding thetherapeutic factor to a patient. For instance, if multiple surgerieshave been performed, the patient displays low tolerance to anesthetic,or if other ocular-related disorders exist, topical administration ofthe therapeutic factor or nucleic acid sequence encoding the therapeuticfactor may be most appropriate. Topical formulations are well known tothose of skill in the art. The use of patches, corneal shields (see,e.g., U.S. Pat. No. 5,185,152), and ophthalmic solutions (see, e.g.,U.S. Pat. No. 5,710,182) and ointments, e.g., eye drops, is also withinthe skill in the art. If desired, the therapeutic factor or the nucleicacid sequence encoding the therapeutic factor can be administerednon-invasively using a needleless injection device, such as theBiojector 2000 Needle-Free Injection Management System® available fromBioject, Inc.

The therapeutic factor or nucleic acid sequence encoding the therapeuticfactor is preferably present in or on a device that allows controlled orsustained release, such as an ocular sponge, meshwork, mechanicalreservoir, or mechanical implant. Implants (see, e.g., U.S. Pat. Nos.4,853,224, 4,997,652, and 5,443,505), devices (see, e.g., U.S. Pat. Nos.4,863,457, 5,098,443, 5,554,187, and 5,725,493), such as an implantabledevice, e.g., a mechanical reservoir, an intraocular device, or anextraocular device with an intraocular conduit, especially an implant ora device comprised of a polymeric composition, are particularly usefulfor ocular administration of the therapeutic factor or nucleic acidsequence encoding the therapeutic factor. The therapeutic factor ornucleic acid sequence encoding the therapeutic factor also can beadministered in the form of a sustained-release formulation (see, e.g.,U.S. Pat. No. 5,378,475) comprising, for example, gelatin, chondroitinsulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate(BHET), or a polylactic-glycolic acid.

Alternatively, the therapeutic factor or nucleic acid sequence encodingthe therapeutic factor can be administered using invasive procedures,such as, for instance, intravitreal injection or subretinal injection,optionally preceded by a vitrectomy. Subretinal injections can beadministered to different compartments of the eye, e.g., the anteriorchamber or posterior chamber. Pharmaceutically acceptable carriers forinjectable compositions are well-known to those of ordinary skill in theart (see Pharmaceutics and Pharmacy Practice, J.B. Lippincott Co.,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4^(th) ed., pages 622-630(1986)).

In some embodiments, it is advantageous to deliver the therapeuticfactor or nucleic acid encoding the therapeutic factor via periocular(e.g., episcleral, sub-tenon, or sub-conjunctival) injection. Forexample, most standard injection techniques require puncturing layers ofthe eye, including the sclera, choroid, retina, etc. To minimize traumato those layers of the eye, the therapeutic factor or nucleic acidencoding the therapeutic factor can be administered into the sub-tenon(i.e., episcleral) space surrounding the scleral portion of the eye. Thesub-tenon space is enclosed by Tenon's capsule, a fibrous sheathencasing the posterior segment of the eye. Puncture of this fibroussheath with an injection device is less traumatic to the layers of theeye responsible for vision. In addition, when the nucleic acid sequenceencoding the therapeutic factor is present in a viral vector,vector-related toxicity to intraocular cells is minimized, as at least aportion of the dose of vector transduces cells of the sub-tenon space,where the nucleic acid sequence encoding therapeutic factor is expressedand the therapeutic factor thereby produced is transported tointraocular cells. Due to the structure of Tenon's capsule, the exposureof non-ocular cells to the therapeutic factor or nucleic acid sequenceencoding the therapeutic factor is limited. Sub-tenon injection alsoallows the administration of a greater volume of therapeutic compositioncompared to that allowed by, for example, subretinal injection.

In most cases, sub-tenon delivery of a composition to the eye involvessurgically opening Tenon's capsule and injecting into the sub-tenonspace using a syringe or cannula. Alternatively, Tenon's capsule isgrasped by the practitioner, not surgically opened, and the therapeuticcomposition is injected into the sub-tenon space using, for example, asyringe. The therapeutic factor or nucleic acid encoding the therapeuticfactor can be administered to other regions of the ocular apparatus suchas, for instance, the ocular muscles, the orbital fascia, the eye lid,the lacrimal apparatus, and the like as is appropriate.

Preferably, the therapeutic factor or nucleic acid sequence encoding thetherapeutic factor is administered via an ophthalmologic instrument fordelivery to a specific region of an eye, e.g., the sub-tenon space. Theuse of a specialized ophthalmologic instrument ensures preciseadministration of the therapeutic factor or the nucleic acid sequenceencoding the therapeutic factor, while minimizing damage to adjacentocular tissue. Delivery of the therapeutic factor or nucleic acidsequence encoding the therapeutic factor to a specific region of the eyealso limits exposure of unaffected cells to the therapeutic factor,thereby reducing the risk of side effects. A preferred ophthalmologicinstrument is a combination of forceps and subretinal needle or sharpbent cannula.

When administering the therapeutic factor or the nucleic acid sequenceencoding the therapeutic factor, appropriate dosage and route ofadministration can be selected to minimize loss of the therapeuticfactor or nucleic acid sequence or inactivation of the therapeuticfactor due to a host's immune system. For example, for contacting ocularcells in vivo, it can be advantageous to administer to a host a nullexpression vector (i.e., an expression vector not comprising the nucleicacid sequence encoding the therapeutic factor) prior to performing theinventive method. Prior administration of a null expression vector canserve to create immunity in the host to the expression vector, therebydecreasing the amount of therapeutic vector cleared by the immunesystem. The therapeutic factor, itself, can be manipulated to maskimmunogenic epitopes or co-administered with an immunosuppressant.

The dose of therapeutic factor or nucleic acid sequence encoding thetherapeutic factor administered to an animal, particularly a human, inaccordance with the invention should be sufficient to effect the desiredresponse (selective induction of apoptosis in endothelial cellsassociated with neovascularization in the animal over a reasonable timeframe). Dosage will depend upon a variety of factors, including the age,species, the pathology in question, and condition or disease state.Dosage also depends on the therapeutic factor, as well as the amount ofocular tissue about to be affected or actually affected by theocular-related disease. The size of the dose also will be determined bythe route, timing, and frequency of administration as well as theexistence, nature, and extent of any adverse side effects that mightaccompany the administration of a particular therapeutic factor ornucleic acid sequence encoding the therapeutic factor, and the desiredphysiological effect. It will be appreciated by one of ordinary skill inthe art that various conditions or disease states, in particular,chronic conditions or disease states, may require prolonged treatmentinvolving multiple administrations.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.When administering a peptide, preferably from about 0.5 mg to about 6 mgof therapeutic factor, more preferably about 2 mg to about 6 mg oftherapeutic factor, is administered per eye. More preferably, from about3 mg to about 5 mg peptide is administered per eye. Most preferably, 4mg peptide is administered per eye. When using a viral vector,preferably about 10⁶ viral particles to about 10¹² viral particles aredelivered to the eye. In other words, a pharmaceutical composition canbe administered that comprises a viral vector concentration of fromabout 10⁶ particles/ml to about 10¹² particles/ml (including allintegers within the range of about 10⁶ particles/ml to about 10¹²particles/ml, e.g., 10⁷ particles/ml, 10⁸ particles/ml, 10⁹particles/ml, 10¹⁰ particles/ml, and 10¹¹ particles/ml), preferablyfrom about 10¹⁰ particles/ml to about 10¹² particles/ml, and willtypically involve the intraocular administration of from about 0.1 μl toabout 100 μl of such a pharmaceutical composition per eye. When thenucleic acid sequence is a plasmid, preferably about 0.5 ng to about1000 μg of DNA is administered per eye. More preferably, about 0.1 μg toabout 500 μg is administered per eye, even more preferably about 1 μg toabout 100 μg of DNA is administered per eye. Most preferably, about 50μg of DNA is administered per eye. Of course, other routes ofadministration may require smaller or larger doses to achieve atherapeutic effect. Any necessary variations in dosages and routes ofadministration can be determined by the ordinarily skilled artisan usingroutine techniques known in the art.

In some embodiments, it is advantageous to administer two or more (i.e.,multiple) doses of the therapeutic factor or nucleic acid sequenceencoding the therapeutic factor. The inventive method provides formultiple applications of the therapeutic factor to selectively induceapoptosis of endothelial cells associated with neovascularization,thereby prophylactically or therapeutically treating ocularneovascularization. For example, at least two applications of anexpression vector comprising an exogenous nucleic acid, e.g., a nucleicacid sequence encoding the therapeutic factor, can be administered tothe same eye. Preferably, the multiple doses are administered whileretaining therapeutic factor concentrations above background levels.Also preferably, the ocular cell is contacted with two applications ormore of the therapeutic factor or nucleic acid sequence encoding thetherapeutic factor via direct administration to the eye within about 30days or more. More preferably, two or more applications are administeredto ocular cells of the same eye within about 90 days or more. However,three, four, five, six, or more doses can be administered in any timeframe (e.g., 2, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 85, ormore days between doses) so long as ocular neovascularization isinhibited or ameliorated.

It also will be appreciated by one skilled in the art that a nucleicacid sequence encoding the therapeutic factor can be introduced ex vivointo ocular cells, previously removed from a given animal, in particulara human. Likewise, cells can be exposed to the therapeutic factor exvivo, although this is less preferred. Such transduced autologous orhomologous host cells, reintroduced into the animal or human, willexpress directly the therapeutic factor in vivo. One ex vivo therapeuticoption involves the encapsidation of infected ocular cells into abiocompatible capsule, which can be implanted in the eye. Such cellsneed not be isolated from the patient, but can instead be isolated fromanother individual and implanted into the patient.

It will be appreciated that an expression vector comprising the nucleicacid sequence encoding the therapeutic factor, preferably an adenoviralvector comprising the nucleic acid sequence, can comprise multiplenucleic acid sequences encoding the therapeutic factor. For example, theexpression vector can comprise multiple copies of the PEDF codingsequence, each copy operably linked to a different promoter or toidentical promoters.

In addition, the nucleic acid sequence encoding the therapeutic factorcan further comprise one or more other transgenes. By “transgene” ismeant any nucleic acid that can be expressed in a cell. Desirably, theexpression of the transgene is beneficial, e.g., prophylactically ortherapeutically beneficial, to the ocular cell or eye. If the transgeneconfers a prophylactic or therapeutic benefit to the cell, the transgenecan exert its effect at the level of RNA or protein. For example, thetransgene can encode a peptide other than the therapeutic factor thatcan be employed in the treatment or study of a disorder, e.g., anocular-related disorder. Alternatively, the transgene can encode anantisense molecule, a ribozyme, a protein that affects splicing or 3′processing (e.g., polyadenylation), or a protein that affects the levelof expression of another gene within the cell (i.e., where geneexpression is broadly considered to include all steps from initiation oftranscription through production of a process protein), such as bymediating an altered rate of mRNA accumulation or transport or analteration in post-transcriptional regulation. The transgene can encodea chimeric peptide for combination treatment of an ocular-relateddisorder. As discussed herein, different promoters have dissimilarlevels and patterns of activity. One of ordinary skill in the art willappreciate the freedom to dictate the expression of different codingsequences through the use of multiple promoters. Alternatively, themultiple coding sequences can be operably linked to the same promoter toform a polycistronic element. The polycistronic element is transcribedinto a single mRNA molecule when transduced into the ocular cell.Translation of the mRNA molecule is initiated at each coding sequence,thereby producing the multiple, separate peptides simultaneously.

An expression vector comprising the nucleic acid sequence encoding thetherapeutic factor can comprise an additional therapeutic nucleic acidsequence, such as a nucleic acid sequence encoding a vessel maturationfactor. Many ocular disorders involve leakage of blood products throughvessels, which can cloud vision and induce an immune response within thelayers of the eye. Vessel maturation factors reduce the amount ofvascular leakage and, therefore, are useful in treating, for example,exudative ocular disorders. Vessel maturation factors include, but arenot limited to, angiopoietins (Ang, e.g., Ang-1 and Ang-2), tumornecrosis factor-alpha (TNF-α), midkine (MK), COUP-TFII, andheparin-binding neurotrophic factor (HBNF, also known as heparin bindinggrowth factor). A nucleotide sequence encoding an immunosuppressor alsocan be incorporated into the expression vector to reduce anyinappropriate immune response within the eye as a result of anocular-related disorder or the administration of the expression vector.

One or more additional nucleic acid sequences encoding ananti-angiogenic substance other than the therapeutic factor can beco-administered. As set forth above, an anti-angiogenic substance is anybiological factor that prevents or ameliorates neovascularization. Oneof ordinary skill in the art will understand that the anti-angiogenicsubstance can effect partial or complete prevention and amelioration ofangiogenesis to achieve a therapeutic effect. An anti-angiogenicsubstance includes, for instance, an anti-angiogenic factor, ananti-sense molecule specific for an angiogenic factor, a ribozyme, areceptor for an angiogenic factor, and an antibody that binds a receptorfor an angiogenic factor.

A nucleic acid sequence encoding marker protein, such as greenfluorescent protein or luciferase, can be present in an expressionvector. Such marker proteins are useful in vector construction anddetermining vector migration. Marker proteins also can be used todetermine points of injection or treated ocular tissues in order toefficiently space injections of a nucleic acid sequence or therapeuticfactor to provide a widespread area of treatment, if desired.Alternatively, a nucleic acid sequence encoding a selection factor,which also is useful in vector construction protocols, can be part ofthe expression vector.

It should be appreciated that any of the therapeutic factors or nucleicacid sequences encoding therapeutic factors described herein can bealtered from their native form to increase their therapeutic effect. Forexample, a cytoplasmic form of a therapeutic nucleic acid can beconverted to a secreted form by incorporating a signal peptide into theencoded gene product. In addition, the therapeutic factor can bedesigned to be taken up by neighboring cells by fusion of the peptidewith VP22. This allows an ocular cell comprising the therapeutic nucleicacid sequence to have a therapeutic effect on a number of ocular cellswithin the mammal. In other words, to contact an endothelial cellassociated with neovascularization, a nucleic acid sequence encoding thetherapeutic factor can transduce a cell in the vicinity of theneovascular process. Upon expression, a secretable therapeutic factorcan be released into the environment of the target cell to exert itstherapeutic effect.

The inventive method also can involve the co-administration of otherpharmaceutically active compounds. By “co-administration” is meantadministration before, concurrently with, e.g., in combination with thetherapeutic factor or nucleic acid sequence encoding the therapeuticfactor in the same formulation or in separate formulations, or afteradministration of the nucleic acid sequence encoding the therapeuticfactor as described above. For example, factors that controlinflammation, such as ibuprofen or steroids, can be co-administered toreduce swelling and inflammation associated with intraocularadministration of the nucleic acid sequence encoding the therapeuticfactor. Immunosuppressive agents can be co-administered to reduceinappropriate immune responses related to an ocular disorder or thepractice of the inventive method. Anti-angiogenic factors, such assoluble growth factor receptors, growth factor antagonists, e.g.,angiotensin, and the like can also be co-administered, as well asneurotrophic factors. Similarly, vitamins and minerals, anti-oxidants,and micronutrients can be co-administered. Antibiotics, i.e.,microbicides and fungicides, can be co-administered to reduce the riskof infection associated with ocular procedures and some ocular-relateddisorders.

While the invention is particularly suited for the treatment ofdisorders involving angiogenesis in the eye, it will be appreciated thatneovascularization is linked to other disorders associated with otherregions of the body. Indeed, as most angiogenesis-related diseasesinvolve uncontrolled or abnormal growth of new blood vessels, thetherapeutic factor or nucleic acid sequence encoding the therapeuticfactor is appropriate for use in the prophylactic or therapeutictreatment of neovascularization-related disorders in other tissues.Accordingly, the invention further comprises a method ofprophylactically or therapeutically treating a tissue for abnormal oruncontrolled neovascularization (i.e., neovascularization having adetrimental effect), wherein the method comprises directly administeringa therapeutic factor or a nucleic acid sequence encoding a therapeuticfactor, which is expressed to produce the therapeutic factor, to atarget tissue to selectively induce apoptosis of endothelial cellsassociated with neovascularization of the target tissue, such thatneovascularization is treated prophylactically or therapeutically.

For example, the therapeutic factor or the nucleic acid sequenceencoding the therapeutic factor can be delivered to tissues including,for example, muscle tissue, joint tissue, skin, and tumor tissue totherapeutically or prophylactically treat neovascularization. Forexample, skin disorders including psoriasis, scleroderma, and hair lossinvolve neovascular complications. Some forms of arthritis stem fromuncontrolled angiogenesis in the joints. In addition, tumor formationand growth are dependent, in part, on the formation of new blood vesselsto deliver nutrients and oxygen to the tumor. These ailments can becontrolled by treating or inhibiting, at least in part, vascularproliferation.

The dosages of therapeutic factor or nucleic acid sequence encoding thetherapeutic factor, compositions, formulations, and other considerationsdescribed above are appropriate for the prophylactic or therapeutictreatment of neovascularization-related disorders in regions of the bodyother than the eye. Local or systemic delivery of the therapeutic factoror nucleic acid sequence encoding the therapeutic factor can beaccomplished by administration comprising application or instillation ofthe formulation into body cavities, inhalation or insufflation of anaerosol, or by parenteral introduction, comprising intramuscular,intravenous, peritoneal, subcutaneous, intraarterial, intraocular, andintradermal administration, as well as topical administration. However,direct administration involving injection or topical applicationdirectly to the target tissue wherein neovascularization is to bemodulated as described in, for example, International Patent ApplicationWO 98/32859, is most preferred. Of course, the routes of administrationdiscussed herein are merely exemplary. The present inventive methods arenot dependent on the particular route of administration or doseadministered.

EXAMPLES

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example illustrates a preferred method of obtaining expression of afactor comprising both anti-angiogenic and neurotrophic activity from anadenoviral vector in in vivo retina.

An adenoviral vector deficient in one or more essential gene functionsof the E1, E3, and E4 regions of the adenoviral genome and comprising aPEDF gene (AdPEDF) is preferably constructed as set forth inInternational Patent Application WO 99/15686 (McVey et al.). However,the method of the invention is not dependent on the method of vectorconstruction employed, and previously described methods of vectorconstruction are also suitable.

Several in vivo models of ocular neovascularization are available.Neovascularization of the retina is obtained in, for example, neonatalanimals, i.e., neonatal mice, by exposing the mice to hypoxic conditionsshortly after birth. Several days later, the neonatal mice are exposedto standard atmospheric conditions, resulting in ischemia-inducedneovascularization of the retina.

AdPEDF is administered to the right eye of at least 12 day old miceanesthetized with, for example, ketamine or a combination of ketamineand xylazine via intravitreal injection. Injections are performed byforming an entrance site in the posterior portion of the eye andadministering approximately 0.1-5.0 μl of a pharmaceutical compositioncomprising AdPEDF. In most instances, an injection of the expressionvector will be administered to only one eye, while the remaining eyeserves as a control. The mice are sacrificed at various time pointsafter administration of the pharmaceutical composition to determine theextent and duration of PEDF expression in the retina. The right and lefteyes of each animal are enucleated and either fixed for histologicalanalysis or prepared for PEDF expression analysis. Detection of PEDFDNA, PEDF RNA, or PEDF protein can be accomplished using methods wellknown in the art, such as PCR and blotting techniques (see, for example,Sambrook et al., supra).

To determine the effect of PEDF on neovascularization in vivo in, forexample, a human, indirect ophthalmoscopy of the retina is ideal.Stereophotographs are useful in detecting extensive neovascularization,but not appropriate for detecting subtle lesions. Apoptosis ofendothelial cells can be detected using the TUNEL assay. Comparision ofthe level of apopotosis in cells associated with new blood vessel growthcan be compared to the level of apoptosis (if any) in cells associatedwith pre-existing blood vessels.

Example 2

This example demonstrates the utility of adenoviral vectors to delivermultiple doses of an exogenous nucleic acid to the eye.

Adenoviral vectors comprising the luciferase gene (Ad.L) or controladenoviral vectors comprising no transgene (Ad.null) were injected intothe intravitreal space of C57BL6 mouse eyes (Day 0). One day followinginjection of the adenoviral vectors (Day 1), eyes infected with Ad.Lwere enucleated and frozen (1^(st) administration). The eyes infectedwith Ad.null were divided into three groups. In Group I, Ad.L vectorswere injected into the intravitreal space of the eye at Day 14 (fourteendays following the initial dose of Ad.null). Group I eyes wereenucleated and frozen the day following the second administration ofadenoviral vectors (Day 15, 2^(nd) administration). Group II eyes wereinjected intravitreally with Ad.null at Day 14, and injectedintravitreally with Ad.L vectors four weeks following the initialinjection with Ad.null (Day 28, 3^(rd) administration). The eyes werethen enucleated and frozen the day after the third administration ofadenoviral vector. Group III eyes were injected intravitreally withAd.null at Day 14 and Day 28, and injected with Ad.L vectors six weeksfollowing the initial injection with Ad.null (Day 42, 4^(th)administration). The eyes were then enucleated and frozen the day afterthe fourth administration of adenoviral vector. Luciferase assays wereperformed on the eye samples to determine the efficiency of infectionand gene expression associated with multiple dosing of the vectors.

Luciferase expression in ocular cells after the 1^(st) and 2^(nd)administration of adenoviral vector was substantially equivalent. Inother words, no loss of gene expression was detected following twoadministrations of the gene transfer vector. Gene expression from the3^(rd) administration of adenoviral vector was between 10- and 100-foldreduced compared to gene expression from the 1^(st) administration andthe 2^(nd) administration, but was still above background levels (e.g.,as detected in cells transduced with Ad.null). Gene expression from the4^(th) administration of adenoviral vector was reduced approximately 3-to 10-fold compared to the gene expression observed following the 3^(rd)administration. However, the level of gene expression following the4^(th) administration was above background levels.

This example demonstrates the feasibility of performing multipleapplications of adenoviral vectors to the eye in order to obtainexpression of an exogenous nucleic acid in ocular cells.

Example 3

This example demonstrates the ability of an expression vector comprisinga nucleic acid sequence encoding a factor comprising bothanti-angiogenic and neurotrophic properties to inhibit choroidalneovascularization (CNV).

Replication-deficient (E1-/E3-deficient) adenoviral vectors (AdPEDF.10)comprising the coding sequence for PEDF operably linked to the CMVimmediate early promoter were constructed using standard techniques. Anull version of the vector (AdNull.10), which did not comprise the PEDFcoding sequence, was also constructed.

Adult C57BL/6 mice were injected intravitreously with AdNull.10 orAdPEDF.10 using a Harvard pump microinjection apparatus and pulled glassmicropipettes. Each eye was injected intravitreously with 1 μl ofvehicle containing 10⁹ particles of virus. Alternatively, each eye wasinjected subretinally with 10⁸ particles of virus suspended in 1 μl ofvehicle. Five days post-injection, mice were anesthetized with ketaminehydrochloride (100 mg/kg body weight). Topicamide (1%) was utilized todilate the pupils prior to rupture of Bruch's membrane by diode laserphotocoagulation. Rupture of Bruch's membrane is known to induceneovascularization of the choroid.

Fourteen days following laser-induced rupture of Bruch's membrane,choroidal flat mounts (described in Edelman et al., Invest. OphthalmolVis. Sci., 41, S834 (2000)) were prepared to observe the degree ofneovascularization of the choroidal membrane. Briefly, eyes were removedfrom the subjects and fixed in phosphate-buffered formalin. The cornea,lens, and retina were removed from the eyecup, and the eyecup wasflat-mounted. Flat mounts were then examined by fluorescence microscopyand images were digitized using a 3 color CCD video camera (IK-TU40A,Toshiba, Tokyo, Japan) for computer image analysis.

Large areas of neovascularization were observed in uninjected eyes andeyes receiving AdNull.10. Eyes injected with AdPEDF.10 subretinally orintravitreously showed smaller regions of neovascularization, ascompared to the controls, using computerized image analysis.

These results illustrate the ability of the inventive method to inhibitocular neovascularization, particularly choroidal neovascularization(CNV), in a clinically animal relevant model.

Example 4

This example demonstrates the ability of an expression vector comprisinga nucleic acid sequence encoding a factor comprising bothanti-angiogenic and neurotrophic properties to inhibit ischemia-inducedretinal neovascularization.

Replication-deficient adenoviral vectors comprising the coding sequencefor PEDF operably linked to the CMV immediate early promoter wereconstructed using standard techniques. E1-/E3-/E4-deficient vectorsencoding PEDF (AdPEDF.11) and a null version of the vector (AdNull.11),which did not comprise the PEDF coding sequence, were constructed.

Ischemic retinopathy was produced in adult C57BL/6 mice as previouslydescribed (see, for example, Smith et al., Invest. Ophthalmol. Vis.Sci., 35, 101 (1994)). In particular, seven day old mice were exposed toan atmosphere of 75±3% oxygen for five days. When the mice were ten daysold, i.e., after three days exposure to the aforementioned oxygenatmosphere, the mice were injected intravitreously with 10⁹ particles ofAdPEDF.11 or AdNull.11, returned to the oxygen atmosphere for two moredays, and then returned to room atmosphere. When the mice were seventeendays old, i.e., five days later, the mice were sacrificed, and theireyes were rapidly removed and frozen in optimum cutting temperatureembedding compound (OCT; Miles Diagnostics, Elkhart, Ind.).

To detect neovascularization, the eyes were sectioned on slides andhistochemically stained with biotinylated griffonia simplicifolia lectinB4 (GSA, Vector Laboratories, Burlingame, Calif.). The slides wereincubated in methanol/H₂O₂ for 10 minutes at 4° C., washed with 0.05 MTris-buffered saline, pH 7.6 (TBS), and incubated for 30 minutes in 10%normal porcine serum. The slides were then incubated for two hours withbiotinylated GSA, rinsed with TBS, and incubated with avidin-coupledalkaline phosphatase (Vector Laboratories) for 45 minutes. After a 10minute wash with TBS, the slides were incubated with Histomark Red.GSA-stained, 10 μm serial sections were examined using an Axioskopmicroscope. Images were digitized using a 3 color CCD video camera(IK-TU40A, Toshiba, Tokyo, Japan) for computer image analysis.

Extensive retinal neovascularization was detected in eyes not injectedwith any expression vector. Eyes injected with AdNull.11 showed lessneovascularization than uninjected eyes, but significantly moreneovascularization of the retina than eyes injected with AdPEDF.11. Eyesinjected with AdPEDF.11 comprised the least amount ofneovascularization.

These results clearly demonstrate the ability of the inventive method toinhibit an ocular-related disorder, particularly ischemia-inducedretinal neovascularization, in a clinically relevant animal model.

Example 5

This example illustrates the ability of the inventive method to promoteregression of ocular neovascularization. This example furtherdemonstrates the induction of apoptosis in endothelial cells associatedwith neovascularization by a therapeutic factor with no damage toexisting vasculature.

AdPEDF.11, i.e., E1-/E3-/E4-deficient adenoviral vectors comprising thecoding sequences for PEDF operably linked to the CMV immediate earlypromoter were constructed as previously described in Example 4.AdNull.11 adenoviral vectors that do not express transgene products alsowere constructed as described in Example 4.

The effect of a therapeutic factor, PEDF, on CNV induced by exposure ofa mouse eye to laser was examined. Briefly, adult C57BL/6 mice wereanesthetized with ketamine hydrochloride (100 mg/kg body weight), pupilswere dilated with 1% tropicamide, and diode laser photocoagulation wasused to rupture Bruch's membrane at 3 locations in each eye of eachmouse as previously described herein. Laser photocoagulation (532 nmwavelength, 100 μm spot size, 0.1 seconds duration, and 120 mWintensity) was delivered using the slit lamp delivery system, and ahand-held cover slide as a contact lens. Bums were performed in the 9,12, and 3 o'clock positions 2-3 disc diameters from the optic nerve.Production of a vaporization bubble at the time of laser, whichindicates rupture of Bruch's membrane, is an important factor inobtaining CNV, so only burns in which a bubble was produced wereincluded in the study.

Two weeks after rupture of Bruch's membrane, a cohort of mice wassacrificed, and the baseline amount of choroidal neovascularization wasmeasured at each rupture site as described below. The remainder of themice received an intravitreous injection of 10⁹ particles or asubretinal injection of 10⁸ particles of AdNull.11 or AdPEDF.11 in eacheye. Intravitreous injections were done with a Harvard pumpmicroinjection apparatus and pulled glass micropipets. Each micropipetwas calibrated to deliver 1 μl of vehicle containing 10⁹ or 10⁸particles upon depression of a foot switch. The mice were anesthetized,pupils were dilated, and under a dissecting microscope, the sharpenedtip of the micropipet was passed through the sclera just behind thelimbus into the vitreous cavity, and the foot switch was depressed.Subretinal injections were performed using a condensing lens system onthe dissecting microscope, which allowed visualization of the retinaduring the injection. The pipet tip was passed through the scleraposterior to the limbus and was positioned just above the retina.Depression of the foot switch caused the jet of injection fluid topenetrate the retina, resulting in fairly uniform blebs that confirmedthat the vector had been deposited in the subretinal space.

To detect choroidal neovascularization, the area of choroidalneovascularization at each rupture site was measured in choroidal flatmounts ten days after vector injection. Mice were anesthetized andperfused with 1 ml of phosphate-buffered saline containing 50 mg/ml offluorescein-labeled dextran (2×10⁶ average MW, Sigma, St. Louis, Mo.).The eyes were removed and fixed for 1 hour in 10% phosphate-bufferedformalin. The cornea and lens were removed, and the entire retina wascarefully dissected from the eyecup. Radial cuts (4-7 cuts, average 5cuts) were made from the edge to the equator, and the eyecup was flatmounted in Aquamount with the sclera facing down. Flat mounts wereexamined by fluorescence microscopy on an Axioskop microscope (Zeiss,Thornwood, N.Y.), and images were digitized using a 3 color CCD videocamera (IK-TU40A, Toshiba, Tokyo, Japan) and a frame grabber. Image-ProPlus software (Media Cybernetics, Silver Spring, Md.) was used tomeasure the total area of hyperfluorescence associated with each burn,corresponding to the total fibrovascular scar. The areas within each eyewere averaged to give one experimental value, and mean values werecalculated for each treatment group and compared by Student's unpairedt-test.

Fourteen or 24 days after laser treatment in adult C57BL/6 mice, therewas extensive choroidal neovascularization at sites of rupture ofBruch's membrane. Mice that received an intravitreous injection of 10⁹particles of AdNull.11 fourteen days after laser treatment, and weresacrificed 10 days later, had large areas of choroidalneovascularization at the sites of rupture of Bruch's membrane thatlooked very similar to those seen in uninjected mice. The same was truefor mice that received a subretinal injection of 10⁸ particles ofAdNull.11 fourteen days after laser treatment and were sacrificed 10days later. This was true regardless of whether the rupture site wasoutside the region of retina that was detached by the subretinalinjection or within the area of the bleb.

In contrast, mice that received an intravitreous injection of 10⁹particles of AdPEDF.11 fourteen days after laser treatment, and weresacrificed 10 days later, had smaller areas of choroidalneovascularization than AdNull.11-injected or uninjected mice. Moreover,the morphology of the hyperfluorescent area was unusual in that theborders were irregular and there was prominent surroundinghyperpigmentation. The appearance is consistent with regression ofchoroidal neovascularization, leaving hyperpigmentation in the region inwhich involution of neovascularization occurred.

Hyperpigmentation surrounding an irregular area of hyperfluorescence wasnot the only morphology that suggested regression in treated mice. In alesion detected 24 days after rupture of Bruch's membrane in a mousethat received a subretinal injection of 10⁸ particles of AdPEDF.11fourteen days after laser treatment, the area of hyperfluorescence issmaller than those seen in AdNull.11-injected or uninjected mice, and itconsists of a few relatively large vessels and no small vessels. Apossible explanation is that the small vessels regressed, leaving only afew larger vessels. This lesion was located within the subretinal blebcaused by the subretinal injection of AdPEDF.11. A second lesion wasstudied from a mouse that was given a subretinal injection of 10⁸particles of AdPEDF.11 fourteen days after laser treatment, but therupture site was outside the region of the bleb. Compared to lesions inAd.Null-injected or uninjected eyes, the second lesion was smaller andhad a morphology consisting of a small region of hyperfluorescencesurrounded by hyperpigmentation, similar to that of lesions seen in manyeyes that received an intravitreous injection of AdPEDF.11.

Measurement of the areas of choroidal neovascularization at rupturesites showed that eyes that received either an intravitreous orsubretinal injection of AdPEDF.11 on day 14 after laser treatment hadsignificantly less neovascularization on day 24 than eyes injected withAdNull.11 or uninjected eyes. Eyes that received a subretinal injectionof AdPEDF.11 also had significantly less neovascularization than thebaseline amount seen at day 14, suggesting that regression of choroidalneovascularization had occurred.

Apoptotic cells were detected by TdT-dUTP terminal nick end-labeling(TUNEL). Ten days after the intraocular injection of vector, and 24 daysafter rupture of Bruch's membrane, the eyes were rapidly removed andfrozen in optimum cutting temperature embedding compound (OCT; MilesDiagnostics, Elkhart, Ind.). Frozen serial sections (10 μm) were cutthrough the entire extent of each burn. Sections were fixed in 4%paraformaldehyde for 20 minutes at room temperature and stained with thein situ cell death detection kit (Roche Diagnostics, Indianapolis, Ind.)according to the manufacturer's instructions. The sections were alsohistochemically stained with biotinylated Griffonia simplicifolia lectinB4 (GSA, Vector Laboratories, Burlingame, Calif.) which selectivelybinds vascular cells. After TUNEL staining, coverslips were removed, andthe slides were incubated in methanol/H₂O₂ for 10 minutes at 4° C.,washed with 0.05 M Tris-buffered saline, pH 7.6 (TBS), and incubated for30 minutes in 10% normal porcine serum. Slides were incubated 2 hours atroom temperature with biotinylated GSA, and after rinsing with 0.05MTBS, they were incubated with avidin coupled to alkaline phosphatase(Vector Laboratories) for 30 minutes at room temperature. After beingwashed for 10 minutes with 0.05 M TBS, slides were incubated withHistomark Red (Kirkegaard and Perry) to give a red reaction product thatis distinguishable from melanin. Some slides were counterstained withContrast Blue (Kirkegaard and Perry).

Fourteen days after laser-induced rupture of Bruch's membrane, micereceived no injection, an intravitreous injection of 10⁹ particles ofAdNull.11 or AdPEDF.11, or a subretinal injection of 10⁸ particles ofAdNull.11 or AdPEDF.11. Ten days later, the mice were sacrificed, andocular sections were labeled with GSA lectin. TUNEL staining was done onadjacent sections, or sections were double-labeled with GSA and TUNEL.Uninjected eyes showed a few TUNEL-stained cells in the retina overlyinglaser-induced choroidal neovascularization, but none within thechoroidal neovascularization. The same was true for eyes that received asubretinal injection of vehicle alone or an intravitreous injection ofAdNull.11. In contrast, TUNEL staining in eyes that received anintravitreous injection of AdPEDF.11 showed a few labeled cells withinchoroidal neovascular lesions, and eyes that received a subretinalinjection of AdPEDF.11 showed numerous labeled cells in choroidalneovascular lesions that also stained with GSA, indicating that theywere dying vascular cells. There was also prominent TUNEL staining ofphotoreceptor cells in regions of retina that had been detached bysubretinal injection of AdPEDF.11. Eyes that had received subretinalinjection of AdNull.11 also showed prominent TUNEL labeling ofphotoreceptors in areas of retina that had been detached by theinjection, but no staining of cells in choroidal neovascular lesions.These results suggest that the death of retinal neurons is attributableto the adenoviral vector, and apoptosis of vascular cells in choroidalneovascular lesions is due to PEDF. In AdPEDF.11-injected eyes, manyTUNEL-stained cells within the choroidal neovascularization and thefeeder vessels from the underlying choroid were observed, but no TUNELstaining of the retinal vascular cells which were not participating inthe neovascularization was detected. Also, choroidal vascular cells inareas not underlying choroidal neovascularization showed no TUNELstaining.

These results demonstrate that direct administration of a nucleic acidsequence encoding a therapeutic factor, which selectively inducesapoptosis in endothelial cells associated with neovascularization,therapeutically treats neovascularization in a clinically-relevantanimal model.

Example 6

This example illustrates the ability of the inventive method to promoteregression of retinal neovascularization.

AdPEDF.11, i.e., E1-/E3-/E4-deficient adenoviral vectors comprising thecoding sequences for PEDF operably linked to the cytomegalovirus (CMV)immediate early promoter, were constructed as previously described inExample 4. AdNull.11 adenoviral vectors that do not express transgeneproducts also were constructed as described in Example.

Transgenic mice with VEGF expression driven by the rhodopsin promoter(rho/VEGF mice) develop subretinal neovascularization due to expressionof VEGF in photoreceptors beginning at about six days after birth and,therefore, present an ideal model for determining the effect of atherapeutic factor, e.g., PEDF, on retinal neovascularization. At 21days after birth, a cohort of transgene-positive mice was sacrificed,and the amount of neovascularization in each retina was measured asdescribed below. The remainder of the mice received no injection or anintravitreous injection of 10⁹ particles of AdNull.11 or AdPEDF.11. At28 days after birth, the mice were sacrificed and the amount ofsubretinal neovascularization was quantified as previously described.Briefly, mice were anesthetized and perfuised with 1 ml ofphosphate-buffered saline containing 50 mg/ml of fluorescein-labeleddextran (2×10⁶ average MW, Sigma, St. Louis, Mo.). The eyes were removedand fixed for 1 hour in 10% phosphate-buffered formalin. The cornea andlens were removed, and the entire retina was carefully dissected fromthe eyecup, radially cut from the edge of the retina to the equator inall 4 quadrants, and flat-mounted in Aquamount with photoreceptorsfacing upward. The retinas were examined by fluorescence microscopy at200× magnification, which provides a narrow depth of field so that, whenfocusing on neovascularization on the outer surface of the retina, theremainder of the retinal vessels are out-of-focus allowing easydelineation of the neovascularization. The outer edge of the retina,which corresponds to the subretinal space in vivo, is easily identifiedand therefore there is standardization of focal plane from slide toslide. Images were digitized using a 3 CCD color video camera and aframe grabber. Image-Pro Plus was used to identify each of the lesions,calculate the number in each retina, delineate the area of each lesion,and delineate the total area of neovascularization per retina.

In rho/VEGF transgenic mice, expression of VEGF in photoreceptors beginsat about six days after birth, increases to a steady-state level byabout 14 days after birth, and continues at that level throughoutadulthood. The total area of subretinal neovascularization per retinasteadily increases between 18 days after birth and 45 days after birth,the oldest age examined. Transgenics that received an intravitreousinjection of 10⁹ particles of AdNull.11 on day 21 after birth, hadextensive neovascularization at 28 days after birth, but it appearedsomewhat less than that seen in uninjected mice 28 days after birth.Transgenics that received an intravitreous injection of 10⁹ particles ofAdPEDF.11 at 21 days after birth, had much less neovascularization thanuninjected mice 28 days after birth; there were RPE cells present, butalmost no hyperfluorescence associated with the RPE cells.Neovascularization had been present and regressed, leaving RPE cellswithout hyperfluorescence in their wake, as supported by quantitativeanalysis. The total area of neovascularization per retina wassignificantly greater at 28 days after birth compared to uninjected miceat 21 days after birth. Mice that received an intravitreous injection of10⁹ particles of AdNull.11 at 21 days after birth and were thensacrificed at 28 days after birth showed less neovascularization thanuninjected mice 28 days after birth, but no significant difference fromuninjected mice at 21 days after birth. This observation reflects thatthe vector itself has some antiangiogenic activity in the transgenicmodel, as was shown in a previous study. However, mice that received anintravitreous injection of 10⁹ particles of AdPEDF.11 at 21 days afterbirth, had less neovascularization at 28 days after birth thanAdNull.11-injected mice at 28 days after birth or uninjected mice at 21days after birth. This confirms that intraocular expression of PEDFcaused regression of subretinal neovascularization in rho/VEGFtransgenic mice.

These results demonstrate that direct administration of a nucleic acidsequence encoding a therapeutic factor, e.g., PEDF, and expression ofthe nucleic acid sequence in the eye, promotes regression ofneovascularization, in particular subretinal neovascularization.

Example 7

This example illustrates that periocular delivery of a nucleic acidencoding a therapeutic factor, e.g., PEDF, inhibits choroidalneovascularization.

Bruch's membrane was ruptured with laser photocoagulation at 3 locationsin each eye of adult C57BL/6 mice. Immediately after laser treatment,mice were given a periocular injection of 3 μl of a compositioncontaining 5×10⁹ particles of AdPEDF.11 (as described in Example 5) inone eye and 5×10⁹ particles of Adnull.11 (as also described in Example5)in the fellow eye. After two weeks, mice were perfused withfluorescein-labeled dextran, and the area of CNV at each rupture sitewas measured on choroidal flat mounts by computerized image analysis asdescribed above.

The mean area of CNV at Bruch's membrane rupture sites was significantlyreduced in eyes given a periocular injection of AdPEDF.11 compared tofellow eyes given a periocular injection of Adnull.11. Thus, a nucleicacid sequence encoding a therapeutic factor delivered periocularlyinhibits neovascularization of the eye.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. Recitation of ranges of values herein are merely intended toserve as a shorthand method of referring individually to each separatevalue falling within the range, unless otherwise indicated herein, andeach separate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations of those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventors expect skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than as specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

1. A method of prophylactically or therapeutically treating choroidalneovascularization, wherein the method comprises intravitreously,subretinally, or periocularly injecting an adenoviral vector comprisinga nucleic acid sequence encoding pigment epithelium-derived factoroperatively linked to a cytomegalovirus (CMV) promoter, which nucleicacid sequence is expressed to produce pigment epithelium-derived factor,to the eye to selectively induce apoptosis of endothelial cellsassociated with neovascularization of the choroid, such that choroidalneovascularization is treated prophylactically or therapeutically. 2.The method of claim 1, wherein apoptosis induced by pigmentepithelium-derived factor in endothelial cells associated withneovascularization of the choroid is at least about 5-times greater thanapoptosis induced by pigment epithelium-derived factor in endothelialcells associated with existing vasculature in the eye.
 3. The method ofclaim 1, wherein apoptosis induced by pigment epithelium-derived factorin endothelial cells associated with neovascularization of the choroidis at least about 10-times greater than apoptosis induced by pigmentepithelium-derived factor in endothelial cells associated with existingvasculature in the eye.
 4. The method of claim 3, wherein apoptosisinduced by pigment epithelium-derived factor in endothelial cellsassociated with neovascularization of the choroid is at least about50-times greater than apoptosis induced by pigment epithelium-derivedfactor in endothelial cells associated with existing vasculature in theeye.
 5. The method of claim 1, wherein pigment epithelium-derived factordoes not affect endothelial cells of existing vasculature.
 6. The methodof claim 3, wherein the existing vasculature is retinal vasculature. 7.The method of claim 1, wherein the adenoviral vector is replicationdeficient.
 8. The method of claim 7, wherein the adenoviral vector isdeficient in one or more gene functions of the E1 region required forviral replication.
 9. The method of claim 8, wherein the adenoviralvector is deficient in one or more gene functions of the E4 regionrequired for viral replication.
 10. The method of claim 7, wherein theadenoviral vector is deficient in one or more gene functions of the E4region required for viral replication.
 11. The method of claim 10,wherein the adenoviral vector is deficient in all gene functionsrequired for viral replication.
 12. The method of claim 1, wherein theadenoviral vector is deficient in one or more gene functions of the E3region of the adenoviral genome.
 13. The method of claim 8, wherein theadenoviral vector is deficient in one or more gene functions of the E3region of the adenoviral genome.
 14. The method of claim 9, wherein theadenoviral vector is deficient in one or more gene functions of the E3region of the adenoviral genome.
 15. The method of claim 10, wherein theadenoviral vector is deficient in one or more gene functions of the E3region of the adenoviral genome.
 16. The method of claim 1, wherein theadenoviral vector is intravitreously injected to the eye.
 17. The methodof claim 16, wherein the adenoviral vector is deficient in one or moregene functions of the E3 region of the adenoviral genome.
 18. The methodof claim 16, wherein the adenoviral vector is replication deficient. 19.The method of claim 18, wherein the adenoviral vector is deficient inone or more gene functions of the E1 region required for viralreplication.
 20. The method of claim 19, wherein the adenoviral vectoris deficient in one or more gene functions of the E4 region required forviral replication.
 21. The method of claim 18, wherein the adenoviralvector is deficient in one or more gene functions of the E4 regionrequired for viral replication.
 22. The method of claim 21, wherein theadenoviral vector is deficient in all gene functions required for viralreplication.
 23. The method of claim 19, wherein the adenoviral vectoris deficient in one or more gene functions of the E3 region of theadenoviral genome.
 24. The method of claim 20, wherein the adenoviralvector is deficient in one or more gene functions of the E3 region ofthe adenoviral genome.
 25. The method of claim 1, wherein the adenoviralvector is subretinally injected to the eye.
 26. The method of claim 25,wherein the adenoviral vector is deficient in one or more gene functionsof the E3 region of the adenoviral genome.
 27. The method of claim 25,wherein the adenoviral vector is replication deficient.
 28. The methodof claim 27, wherein the adenoviral vector is deficient in one or moregene functions of the E1 region required for viral replication.
 29. Themethod of claim 28, wherein the adenoviral vector is deficient in one ormore gene functions of the E4 region required for viral replication. 30.The method of claim 27, wherein the adenoviral vector is deficient inone or more gene functions of the E4 region required for viralreplication.
 31. The method of claim 30, wherein the adenoviral vectoris deficient in all gene functions required for viral replication. 32.The method of claim 28, wherein the adenoviral vector is deficient inone or more gene functions of the E3 region of the adenoviral genome.33. The method of claim 29, wherein the adenoviral vector is deficientin one or more gene functions of the E3 region of the adenoviral genome.34. The method of claim 1, wherein the adenoviral vector is periocularlyinjected to the eye.
 35. The method of claim 34, wherein the adenoviralvector is deficient in one or more gene functions of the E3 region ofthe adenoviral genome.
 36. The method of claim 34, wherein theadenoviral vector is replication deficient.
 37. The method of claim 36,wherein the adenoviral vector is deficient in one or more gene functionsof the E1 region required for viral replication.
 38. The method of claim37, wherein the adenoviral vector is deficient in one or more genefunctions of the E4 region required for viral replication.
 39. Themethod of claim 36, wherein the adenoviral vector is deficient in one ormore gene functions of the E4 region required for viral replication. 40.The method of claim 39, wherein the adenoviral vector is deficient inall gene functions required for viral replication.
 41. The method ofclaim 37, wherein the adenoviral vector is deficient in one or more genefunctions of the E3 region of the adenoviral genome.
 42. The method ofclaim 38, wherein the adenoviral vector is deficient in one or more genefunctions of the E3 region of the adenoviral genome.